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WO2016191839A1 - Procédé d'obtention de vésicules de membrane externe (omv) à partir de la lignée de haemophilus aegyptius 254 (hae254), vésicules de membrane externe ainsi obtenues (omv-hae254) et leur utilisation pour la transfection cellulaire - Google Patents

Procédé d'obtention de vésicules de membrane externe (omv) à partir de la lignée de haemophilus aegyptius 254 (hae254), vésicules de membrane externe ainsi obtenues (omv-hae254) et leur utilisation pour la transfection cellulaire Download PDF

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Publication number
WO2016191839A1
WO2016191839A1 PCT/BR2016/000055 BR2016000055W WO2016191839A1 WO 2016191839 A1 WO2016191839 A1 WO 2016191839A1 BR 2016000055 W BR2016000055 W BR 2016000055W WO 2016191839 A1 WO2016191839 A1 WO 2016191839A1
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WIPO (PCT)
Prior art keywords
omv
hae254
nadh
hemina
outer membrane
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Ceased
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PCT/BR2016/000055
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English (en)
Portuguese (pt)
Inventor
Marcelo LANCELLOTTI
Thais Holtz THEIZEN
Daisy MACHADO
Luciana Maria DE HOLLANDA
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Universidade Estadual de Campinas UNICAMP
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Universidade Estadual de Campinas UNICAMP
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    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • C12R2001/21—Haemophilus

Definitions

  • the present invention relates to the process for obtaining biofermentation of outer membrane vesicles (OMV) from Haemophilus aegyptius 254 (Hae254) lineage, thus obtained outer membrane vesicles (OMV-Hae254) and their use as a facilitator. of the process of cell transfection.
  • OMV outer membrane vesicles
  • OMVs outer membrane vesicles
  • Outer membrane vesicles are evaginations of the outer membrane of bacteria, released into the environment where the bacteria is proliferating, being 10 to 100 nm in diameter. They are made up of all components present on the surface of the bacteria's outer membrane, ie polysaccharides, phospholipids, lipooligosaccharides and mainly proteins.
  • Gram-negative bacteria like all other cells, interact with the environment. These interactions are often performed by the distal effects of the secreted molecules. Secretion is an important aspect of gram-negative bacteria, which offer distinct advantages over materials directly associated with the bacterial cell. Through secretion the bacteria can interact with the environment without expending energy to move. The secreted material is smaller and not viable, so it can influence an environment that is inaccessible to the entire bacterium, either due to its size or restrictions on the bacterial growth present at that site. Secreted materials are typically soluble, however cells belonging to all domains of life have specific mechanisms for secreting material.
  • This envelope is It is formed by two inner and outer membranes, a peptidioglican layer, and the periplasma.
  • the membranes differ in lipid and protein composition.
  • the outer membrane outer monolayer (OM) is composed mainly of lipopolysaccharide (LPS), whereas the inner leaflet and both inner membrane leaflets are composed of phospholipids.
  • LPS lipopolysaccharide
  • the periplasmic space between the two membranes accounts for 7 to 40% of the total cell volume.
  • Periplasma is an oxidizing environment devoid of any energy source known as ATP or NADPH.
  • Within the periplasma is a thin layer of rigid peptidioglican bound to both membranes by membrane-anchored proteins.
  • OMVs outer membrane and periplasm through the production of OMVs, small spherical structures between 20-250 nm in diameter.
  • OMVs are produced when small portions of OM evaginate and detach from the bacterial cell.
  • Soluble proteins are associated with OMVs in the periplasma and externally as adherent material.
  • Such nanoparticles can spread far from the cellular environment and transmit biological functions in the environment and other cells, including playing a role in pathogenesis, quorum sensing, nutrient acquisition and even horizontal gene transfer.
  • OMVs As secreted, insoluble and soluble bacterial surface component complexes, OMVs are ready to perform their biological function of interacting with the environment in which the bacteria is immersed. An important feature of OMVs is that the proteins associated with them exhibit biological activities.
  • OMVs are a delivery and secretion system in a way that enables the diffusion of bacterial products and interaction with the environment. Like other secretion systems, these are mediated by OMVs and are temporally and spatially regulated. However, OMV secretion is distinct from the best studied soluble protein secretion systems. In OMV-mediated secretion, soluble material is released into a complex with and / or surrounded by insoluble material as it may be trapped in the lipid core of the membrane or in the hydrophilic interior of the nanoparticle. On the other hand, the secretory pathways of soluble materials export specific monomers or small protein complexes, and secretion is not accompanied by the release of other cellular materials as in OMV, which comes off with portions even of the bacterial periplasma.
  • a unique feature of OMV secretion is the possibility of secretion of bacterial lipids, membrane proteins, and other compounds. insoluble bacteria cells. Coadherence and coaggregation of bacteria require the presence of functional multivalent adhesin proteins and OVs present multivalent membrane adhesin complexes and can thus satisfy this cellular role and assist in biofilm development.
  • the first step in obtaining an O V is the projection of the bacterial outer membrane toward the extracellular methoid. This implies that such projections are formed in areas where the OM binding proteins and the pepditioglican layer are absent. This may occur if the peptidioglican undergoes antibiotic or autoisine action.
  • adjuvants of microbial origin are licensed and may be used in various experimental groups as adjuvants and drug carriers.
  • Neisseria meningitidis outer membrane vesicle (OMV) is among the newly studied substances of microbial origin, which can be applied as an adjuvant.
  • DEAE-dextran as a cationic polymer. Since this polymer is well associated with exogenous nuclear material, as well as mammalian cells, they endocyte exogenous material.
  • Liposomes Lipid bilayers that form colloidal particles in aqueous environments are called liposomes.
  • Felgner and colleagues developed synthetic cationic lipids.
  • the cationic head of the lipid compound is associated with the negative charge of the nucleic acid phosphate group.
  • liposomes were able to mediate DNA, RNA and protein delivery with high efficiency in cells refractory to calcium phosphate or DEAE-dextran treatment in in vitro and in vivo experiments for both animals and humans.
  • the most common lipid used for gene therapy is positively charged at physiological pH. Often cationic lipid is mixed with neutral lipid such as L-dioleoyl phosphatylethanolamine (DOPE) which increases transfer ability. genic. This is likely due to electrostatic interactions between the negative charge of the nucleic acid and the positive portion of the synthetic lipid head.
  • DOPE L-dioleoyl phosphatylethanolamine
  • genic This is likely due to electrostatic interactions between the negative charge of the nucleic acid and the positive portion of the synthetic lipid head.
  • the positive charge of the liposome / nucleic acid complex binds to the negatively charged plasma membrane generating high transfection rates. Complex entry occurs by endocytosis or plasma membrane fusion via liposome lipids. Following cellular internalization, the complex appears in endosomes and later in the nucleus.
  • Another physical gene delivery methodology is biobalistics. This method is based on the delivery of high speed microprojectiles containing nucleic acids in the target cell and in vivo. Another transfection methodology is as described below.
  • Adenoviral vectors are widely used in gene transfer due to the rapid infectivity of a wide variety of human cells achieving high rates of gene transfer compared to other vectors. They also accommodate large segments of DNA (approximately up to 7.5 kb) to be transferred and are easy to manipulate when gene transfer, this methodology also has its problems. The main problem is the reversal of the inactivated form to the active form which will trigger damage to the host or cell where this methodology was used.
  • Nanotechnology is the science that manipulates atoms, molecules, and supramolecular structures to create and use nanoscale materials, devices, and systems. Thus, given the smallness of their particles, these nanomaterials are easily endocyted by cells. Thus, the use of nanotechnology in biological systems has been called nanobiotechnology. In addition, various nanomaterials, nanodevices, and nanocarriers are used to investigate, treat, and diagnose cancer patients.
  • the present invention advantageously addresses a cell transfection process that utilizes as an exogenous carrier material an outer vesicle membrane obtained from the fermentation of Hae254.
  • OMV-Hae254 presents a simple and efficient alternative for the transfer of exogenous genetic material in in vitro cell culture.
  • the present invention relates to the process of obtaining biofermentation of outer membrane vesicles (OMV) from Haemophilus aegyptius 254 (Hae254) lineage, thus obtained outer membrane vesicles (OMV-Hae254) of the process of cell transfection.
  • OMV outer membrane vesicles
  • the process for obtaining biofermentation of outer membrane vesicles (OMV) comprises the following steps:
  • Figure 1 refers to the curve constructed with the concentrations and absorbances obtained from the standard solution and applying the least squares method to the data to obtain the line equation.
  • Figure 2 is the result of the NG97 cell line MTT and NR Cytotoxicity Assay against different concentrations of OMV-Hae254 after 24 hours.
  • Annex 1 refers to the result of the NG97 Cell Line Transfection Assay (40X Increase), where (A) is the Control without OMV-Hae254, without Effectene, without Pires, without fluorescence; (B) is Control without OMV-Hae254, without Effectene, without pyres, with fluorescence; (C) is Control only with OMV-Hae254 without fluorescence; (D) is Control only with OMV-Hae254 with fluorescence; (E) is Effectene transfection, with pyres, without fluorescence; (F) is Effectene transfection, with pyres, with fluorescence; and, (G) is Transfection of OMV-Hae254 with pIRES without fluorescence (H) is Transfection of OMV-Hae254 with pIRES with fluorescence.
  • the present invention relates to the process for obtaining biofermentation of outer membrane vesicles (OMV) from Haemophilus aegyptius 254 (Hae254) lineage, thus obtained outer membrane vesicles (OMV-Hae254) and their use as a facilitator. of the process of cell transfection.
  • OMV outer membrane vesicles
  • OMV outer membrane vesicles
  • step (d) Cultivate an area of at least 1x10 6 bacteria Haemophilus influenza biotype aegyptius 254 (Hae254) in the medium (AC / NADH / Hemina) obtained from step (b6);
  • the medium was evenly distributed with a gamma-sterile 25 mL serological pipette (Perfecta, China). In each 90x15 plastic petri dish (Cralplast, Brazil) was placed 20 mL.
  • Haemophilus influenza biotype aegyptius 254 (Hae254) strain was cultured using the AC / NADH / Hemina plaque depletion technique as prepared in step (b).
  • the Hae254 petri dish was then incubated in a bacteriological greenhouse (Solab Cient ⁇ fico, Brazil) at 37 ° C for 18 to 24 hours.
  • Step (b) Preparation of the Hae254 Pre-Clause for further production of OMV-254.
  • Step (b) Following bacterial growth in the AC / NADH / Hemina medium (Step (b)), they were removed from the Petri dish by pre-sterilized plastic loop loop scraping (Jet Biofil, USA) approximately 10 to 50 pL of Grown bacteria.
  • the handle with the bacteria was introduced into a Falcon tube (Nerbe plus, Germany) containing 40 mL of BHI / NADH / Hemina liquid medium. The loop was shaken in the middle by circular movements until the bacteria completely detached. The tube was then placed in a bacteriological oven (Solab Cient ⁇ fico, Brazil) for 6 hours at 37 ° C.
  • step (c) The pre-inoculum 10 mL was then added to 1000 mL of BHI / NADH / Hemina medium (step (c)) distributed in a previously autoclaved bioreactor vial (New Brunswick, Bio Flo / Celligen 115, Eppendorf, Brazil).
  • step (e) the tubes containing the biofermented were centrifuged (Sorvall, RT 6000B, USA) for 5 minutes at 4000rpm (3307g).
  • the supernatant was separated into 50 mL Falcon-type tubes for subsequent filtration on a 0.025 ⁇ porosity nitrocellulose filter with a diameter of 142mm. (Millipore, Brazil) with the aid of a compressor at 400 to 600mmHg (NewPump, Sparmax, USA).
  • the OMV - / - / ae254 retained on the filter was solubilized in 1x Phosphate-Saline Buffer (NaCI 0.1369M, KCI 0.00268M, Na 2 HP0 4 0.008M, KH2PO4 0.0 5 ⁇ ) or NaCI 0 0.9% (0.9 g NaCl in 100 mL autoclaved distilled H 2 O) as follows.
  • the filter was removed from the filter apparatus with the aid of 13 cm stainless steel tweezers, 14 cm round nozzle (Professional CE, Brazil) and placed on 12x12 cm plates (Greiner Bio one, Germany). Then, the filter plate, 20 mL or 1x Phosphate Buffer Solution or 0.9% NaCl solution was added. The filter plate and solubilization solution were placed for 30 minutes at 37 ° C in a bacteriological oven (Sorvall, RT 6000B, USA).
  • the NG97 cell line (Astrocytoma type Hl, kindly donated by Prof. Dr. Liana Verinaud) was thawed at room temperature. Then, the entire contents of the cryogenic tube (between 1.0 mL and 1.5 mL) were poured into a cell culture flask with a 0.22 ⁇ tampa filter cap and 25 cm 2 size (NEST, USA) containing 9 mL RPMI 1640 medium (Cultilab, Br), 1 mL (10%) fetal bovine serum (Cultilab, Br) and 100 ⁇ L (1%) of penicillin / streptomycin solution (Cultilab, Br).
  • the cell culture incubator (Sanyo, JP) was incubated at 37 ° C with 5% CO 2 until confluent monolayer formation. While the confluent monolayer did not form the medium containing 9 mL RPMI 1640 medium (Cultilab, Br), 1 mL (10%) fetal bovine serum (Cultilab, Br) and 100 pL (1%) of penicillin / streptomycin solution (Cultilab, Br) was changed every 48 hours and incubated again in an oven at 37 ° C with 5% CO2. In approximately six days (144 hours) the monolayer was formed and the cell ready to be passed on. Cells were twice passaged in 0.22 ⁇ filter-capped bottles of 75 cm 2 size (NEST, USA) prior to use for cytotoxicity and transfection testing. The bottles were trypsinized and distributed into 96-well plates (NEST, USA) for
  • the 24-well or cytotoxicity assay (NEST, USA) for the transfection assay.
  • MTT (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium) 3-bromide to formazan.
  • MTT reduction is mainly catalyzed by mitochondrial dehydrogenases and also by cytoplasm. Therefore, alteration of mitochondrial function may be detected by varying the MTT reduction capacity.
  • optical densities obtained from Step (e) were 1.40 at 12 hours, 1.62 at 16 hours and 1.34 at 20 hours of biofermentation.
  • Table 1 Weight average of 50ml_ of centrifuged pellet in the tube, in each of the steps and hours of the process.
  • Table 2 Quantification of OMV-Hae254 proteins originated at different times in relation to the standard curve.
  • Table 3 Relationship between bacterial sediment mass and OMV-Hae254 production.
  • Figure 2 shows the absence of cytotoxicity effect at different OMV-Hae254 concentrations (0.01 to 10 ⁇ g / mL) within 24 hours.

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Abstract

La présente invention concerne un procédé d'obtention par biofermentation de vésicules de membrane externe (OMV) à partir de la lignée de Haemophilus aegyptius 254 (Hae254), vésicules de membrane externe ainsi obtenues (OMV-Hae254) et leur utilisation pour faciliter le processus de transfection cellulaire.
PCT/BR2016/000055 2015-06-03 2016-05-31 Procédé d'obtention de vésicules de membrane externe (omv) à partir de la lignée de haemophilus aegyptius 254 (hae254), vésicules de membrane externe ainsi obtenues (omv-hae254) et leur utilisation pour la transfection cellulaire Ceased WO2016191839A1 (fr)

Applications Claiming Priority (2)

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BR102015013040-6A BR102015013040B1 (pt) 2015-06-03 2015-06-03 Uso de vesículas de membrana externa (OMV) a partir da linhagem de Haemophilus aegyptius 254 (Hae254)
BRBR1020150130406 2015-06-03

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BR102016028507A2 (pt) * 2016-12-05 2018-06-19 Universidade Estadual De Campinas - Unicamp Vacina para haemophilus aegyptius e uso
BR102017023366A2 (pt) * 2017-10-30 2020-04-28 Univ Estadual Campinas Unicamp vacina anti-zika vírus

Citations (2)

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WO2001089535A1 (fr) * 2000-05-24 2001-11-29 University Of Maryland Biotechnology Institute Procede d'introduction et d'expression de genes dans des cellules animales, et vesicules bacteriennes destinees a etre utilisees dans le cadre dudit procede
US7384645B2 (en) * 2001-12-17 2008-06-10 Health Protection Agency Outer membrane vesicles from gram negative bacteria and use as a vaccine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001089535A1 (fr) * 2000-05-24 2001-11-29 University Of Maryland Biotechnology Institute Procede d'introduction et d'expression de genes dans des cellules animales, et vesicules bacteriennes destinees a etre utilisees dans le cadre dudit procede
US7384645B2 (en) * 2001-12-17 2008-06-10 Health Protection Agency Outer membrane vesicles from gram negative bacteria and use as a vaccine

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BRENNER, D. J. ET AL.: "Biochemical , genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.", J. CLIN. MICROBIOL., vol. 26, no. 8, 1988, pages 1524 - 1534, XP055332108 *
CURY, G. C. ET AL.: "Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever.", BRAZ J MICROBIOL., vol. 45, no. 4, 2014, pages 1449 - 1454, XP055332111 *
CURY, G. C. G.: "Estudo Molecular in vitro da transferência horizontal de genes entre as bactérias Haemophilus influenzae e Neisseria meningitidis.", TESE (DOUTORADO EM BIOLOGIA FUNCIONAL E MOLECULAR, 2013, Campinas, Retrieved from the Internet <URL:http://www.bibliotecadigital.unicamp.br/document> [retrieved on 20160507] *
ROIER, S ET AL.: "Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice.", PLOS ONE., vol. 7, no. 8, 2012, pages e42664, XP055332110 *
WEYANT, R. S. ET AL.: "Purification and characterization of a pilin specific for Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius (H. aegyptius) strains", J CLIN MICROBIOL., vol. 28, no. 4, 1990, pages 756 - 763, XP055332104 *

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