WO2005086870A2 - Nouvelles souches de lactobacille et procede d'utilisation correspondant - Google Patents

Nouvelles souches de lactobacille et procede d'utilisation correspondant Download PDF

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WO2005086870A2
WO2005086870A2 PCT/US2005/007794 US2005007794W WO2005086870A2 WO 2005086870 A2 WO2005086870 A2 WO 2005086870A2 US 2005007794 W US2005007794 W US 2005007794W WO 2005086870 A2 WO2005086870 A2 WO 2005086870A2
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lactobacillus
mbl
ability
hiv
composition
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WO2005086870A3 (fr
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Lin Tao
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The Board Of Trustees Of The University Of Illinois
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/143—Fermentum

Definitions

  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • the present invention provides a method for altering the bacterial composition of the digestive tract or urogenital tract of a mammalian subject comprising administering to the subject bacteria of at least one MBL-positive Lactobacillus strain.
  • the present invention provides a composition comprising MBL-positive Lactobacillus bacteria, the bacteria characterized by the ability to co- aggregate with Saccharomyces cerevisiae and to bind to gpl20.
  • the present invention provides a method of evaluating a Lactobacillus isolate for its suitability for use in methods or compositions of the invention comprising testing the isolate for the ability to co-aggregate with a yeast, bacterium, or virus comprising mannose on its cell wall or envelope, the ability to bind a mannose containing viral coat protein, the ability to bind at least one HIV -target cell, or the ability to block HIV infection.
  • Fig. 1 is an image of co-aggregating mannose binding lectin positive Lactobacillus and yeast.
  • Fig. 2 is a graph showing the results of quantitative binding of various Lactobacillus isolates to immobilized gpl20.
  • Fig. 3 is a graph showing the results of quantitative binding of Lactobacillus fermentum OLB 19a to viral envelop proteins form different clades of HIV and SIV.
  • Fig. 4 is an image showing binding of mannose binding lectin positive Lactobacillus to HIV -target cells.
  • Fig. 5 is a graph showing the number of HIV viruses bound as a function of the Lactobacillus cell concentration.
  • Fig. 6 compares the number of HIV viruses trapped by an MBL-negative Lactobacillus and two MBL-positive Lactobacillus strains.
  • Fig. 7 shows the relative fluorescence of Luciferase-tagged HIV present in a cell culture observed following infection with or without preincubation with a MBL- positive Lactobacillus in various concentrations.
  • Fig. 8 shows the number of HIV virions in cells exposed to HIV in the presence of Lactobacillus OLB43b as a function of exposure time (Fig. 8A) and cell concentration (Fig. 8B).
  • Fig. 9 shows the number of HIV or SIV virions bound by Lactobacillus cells as a function of Lactobacillus strain.
  • Fig. 10 is an image showing the presence or absence of capture by MBL positive lactobaciUi to plain green fluorescent polystyrene beads (Fig. 10A) or gpl20- linked green fluorescent polystyrene beads (Fig. 10B). DETAILED DESCRIPTION
  • Innate immunity is the first line of defense, and includes anatomic barriers, defensive cells, humoral factors, and commensal bacteria.
  • Infants host a large number of commensal bacteria. Infants begin acquiring commensal bacteria during or after birth. It is envisioned that altering the composition of commensal bacteria of an infant's digestive tract may afford protection against infection by HIV or other pathogens. Similarly, altering the composition of commensal bacteria of the vagina may protect against HIV infection or other pathogens.
  • HIV contains mannose in the glycoproteins of its envelope.
  • the presence of mannose in the glycoproteins is fairly constant and relatively insensitive to antigenic shifts in HIV caused by mutational changes.
  • Human mannose-binding lectin (MBL) is a plasma protein that binds to mannose-containing microorganisms, including viruses, bacteria, fungi, and protozoa. Binding of mannose-containing pathogens by MBL facilitates clearance of the pathogen by complement activation and opsonization, and subsequent phagocytosis.
  • human MBL is found principally, if not exclusively, in the plasma, it does not provide infants with a first line of defense at the site of infection, specifically, in the digestive tract.
  • Lactobacillus strains were isolated from saliva of apparently healthy human subjects and tested for the ability to coaggregate with Saccharomyces cerevisae (baker's yeast), a yeast containing mannose. Of the tested strains, nine were found to co-aggregate with S. cerevisae. The addition of mannose at concentrations of 50 mM or 100 mM prevented or reversed Lactobacillus co-aggregation with S. cerevisae.
  • Lactobacillus strains having the ability to co-aggregate with S. cerevisae are referred to herein as MBL-positive Lactobacillus strains.
  • Transposon mutants of a MBL positive Lactobacillus fermentum strain OLB- 19a were created and a putative MBL disruption mutant was selected on the basis of its inability to bind yeast, and designated TM-4A3.
  • the region of the chromosome of TM- 4A3 into which the transposon was inserted has been subcloned and sequencing of that region of the putative MBL disruption mutant is currently under way.
  • the nine Lactobacillus strains capable of co- aggregating S. cerevisae were evaluated for the ability to bind to the HIV envelope glycoproteins gpl20 from strain SF2.
  • Six strains (OLB- 12, OLB- 19a, OLB-24b, OLB- 36b, OLB-43b, and 101S) were found to bind to gpl20.
  • the mutant TM-4A3 does not bind to gpl20.
  • OLB- 19a which was found to bind to gpl20 with a relatively high affinity, was further tested and found to bind to envelope glycoproteins from several different clades of HIV or simian immunodeficiency virus (SIV).
  • the six gpl20-binding strains were assigned to three different species based on 16S rRNA gene analysis as follows: OLB-19a and 101S belong to L. fermentum; OLB-24b belongs to L. gallinarium; and OLB- 12, OLB-36b, and OLB-43b belong to L. delbrueckii subsp. lactis.
  • the glycoprotein of interest was immobilized in the wells of a microtiter plate, the glycoprotein was contacted with cells of a test bacterial strain, unbound cells were removed by washing, bound cells were removed by elution with mannose, and the cells were plated and counted.
  • the HIV glycoprotein was immobilized on a glass slide, contacted with cells of a test bacterial strain, washed and Gram-stained to visually detect bacteria bound to the slide through the glycoprotein. It is envisioned that any suitable means may be used in screening Lactobacillus isolates for the ability to bind to HIV envelope glycoproteins.
  • virus capture ELISA demonstrated that both Lactobacillus OLB- 19a and OLB-43b bind to free HIV-l Ba virus in a dose-dependent manner, and that OLB- 19a binds more free HIV than OLB-43b binds.
  • OLB- 19a was found to bind all HIV target cell types tested, including T and B lymphocytes, monocytes, and cells expressing DC- SIGN. The binding was reversible upon addition of 50 mM mannose.
  • OLB-43b affords greater protection against infection by HIV in a dose-dependent manner.
  • the protective effect may be due to factors that destroy HIV viruses, such as secreted or surface-bound MBL or proteinases.
  • vaginal MBL positive Lactobacillus isolates were characterized with respect to various properties, including yeast binding, gpl20 binding, HIV trapping, and inhibiting the growth of yeast or pathogenic bacteria. It is envisioned that isolates having desirable characteristics may be used to alter the composition of bacteria present in the vagina. This may afford protection against viruses such as HIV, yeast, or pathogen bacteria. The protection may be transient or extended over time, depending on the length of time the MBL positive Lactobacillus strains persist.
  • MBL positive Lactobacillus strain used alone will afford protection against pathogens
  • more than one MBL positive Lactobacillus strain may be used in combination.
  • such combinations may include a Lactobacillus strain or strains having a surface bound MBL and a Lactobacillus strain that secrets MBL.
  • a strain such as OLB- 19a may be used in combination with a strain such as OLB-43b.
  • Oral Lactobacillus strains producing MBL have been shown to reduce the ability of HIV contacted with the strains to infect HIV target cells.
  • the ability of a MBL positive Lactobacillus strain to reduce infectivity of HIV may be associated with its mannose binding activity. It is therefore reasonably expected that the Lactobacillus strains will also afford protection against or reduce colonization of the digestive tract or infection by other mannose or MBL-like adhesin containing organisms, including, but not limited to, pathogenic E.
  • coli Salmonella species, Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae, Candida species, Aspergillus fumigatus, Cryptosoporidium parvum, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, rotaviruses, hepatitis B, hepatitis C, herpes simplex, Nipah (a causal agent of fatal hemorrhagic encephalitis), ⁇ bola, and influenza A and B viruses.
  • Lactobacillus strains described in the Examples are of oral or vaginal origin, it is specifically envisioned that Lactobacillus strains isolated from other sources (e.g., milk) would be useful in the practice of the present invention.
  • Lactobacillus isolates suitable for use in the present invention that may be identified by screening using any suitable assay, including, but not limited to, those described herein.
  • a Lactobacillus isolate can be evaluated for putative MBL activity using coaggregation assays, binding to a mannose-containing glycoprotein, or by binding to yeast.
  • the ability to bind to HIV target cells or to free viruses, or to protect HIV target cells can be assayed as described herein, or using any other suitable assay.
  • Lactobacillus fermentum OLB- 19a and L. delbureckii subsp. lactis. OLB-43b were deposited under the terms of the Budapest treaty with the American Type Culture Collection in Manassas, VA on March 5, 2004 as ATCC accession numbers PTA-5849 and PTA-5850, respectively.
  • the present invention includes derivatives of Lactobacillus fermentum OLB- 19a and L. delbureckii subsp. lactis. OLB-43b.
  • a derivative of a Lactobacillus strain is an organism that is selected, mutagenized, genetically engineered or otherwise obtained from that strain.
  • an MBL positive Lactobacillus strain or strains may be delivered as a probiotic shortly after birth to an infant at risk of orally contracting HIV or other pathogens containing mannose or MBL-like adhesins through breast milk or contaminated water or any other means.
  • Human T-cell lymphotropic virus can also be transmitted from mother to child through breast milk, including colostrum, presumably through T-lymphocytes infected with the virus present in breast milk. It is likely that hepatitis may also be present in breast milk of infected women.
  • the bacteria Prior to administration, the bacteria may be lyophilized and stored in the lyophilized form, and reconstituted just prior to administration with an acceptable carrier, such as sterile water or breast milk, and delivered in an amount effective to establish colonization.
  • the Lactobacillus may be delivered as a symbiotic in conjunction with one or more prebiotics.
  • Prebiotics are carbohydrates that are used probiotic bacterium, but are not substantially used by the host or other bacteria present. Symbiotics are a preferred formulation, because the added prebiotic sugar or sugars can promote the growth and colonization of the probiotics. Because trehalose can be used by many bacteria, it is not suitable as a prebiotic.
  • Glucono-delton-lactone (GDL) can be utilized by OLB 19a, and mannitol can be utilized by OLB43b, but neither can be used by the host and by most other bacteria. Therefore, these two sugars are prebiotics for these lactobaciUi.
  • GDL is a natural constituent of many foods and mannitol is an edible food additive. Both GDL and mannitol are generally regarded as safe (GRAS) by FDA.
  • Agarwal et al. describes the delivery of Lactobacillus rhamnosus strain GG to low birth weight neonates for use as a probiotic.
  • Agarwal et al. describes using twice daily doses of 10 9 organisms for eight days for newborns receiving antibiotics. It is envisioned that fewer doses may be necessary for infants not on antibiotics.
  • Colonization may be evaluated by performing colony counts on stool samples. It is envisioned that it would be preferable to deliver the bacteria to the infant as soon as possible after birth. Infants receiving the bacteria later may require more doses to effectively colonize the digestive tract. An infant may require an additional dose or doses following a course of antibiotics.
  • MBL-producing Lactobacillus strains may be used in a subject of any age to colonize the digestive tract, vagina or rectum and may serve as a barrier to infection by mannose or MBL-like adhesin containing pathogens through those routes. Colonization of the vagina may be especially useful in protecting against infection by HIV, hepatitis B, hepatitis C, herpes simplex, E. coli, Neisseria gonorrhoeae, and Chlamydia trachomatis, or in preventing or treating bacterial vaginosis or vaginal candidiasis.
  • the Lactobacillus may be provided in any suitable form, including, but not limited to, lyophilized form as a powder for direct ingestion or for ingestion following reconstitution, as a powder spray, as nipple cream, in capsule form, as a rectal or vaginal suppository, in feminine hygiene products such as tampons, as a candy-like product, as a nasal spray, or in food, such as yogurt.
  • the Lactobacillus may be provided as a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.
  • the present invention would be suitable for use in economically important species other than humans, including livestock, or in species that serve as reservoirs of disease.
  • diarrhea in cattle caused by mannose or MBL-like adhesin containing pathogens may be reduced by colonizing the gut of cattle with an MBL-positive Lactobacillus.
  • the Lactobacillus strains could be delivered in liquid form by reconstituted lyosphilized bacteria, in a capsule, or mixed with feed.
  • an isolated MBL protein from a Lactobacillus strain to the nasal passages may confer protection against or treat infections acquired through that route or respiratory tract or lungs infections, such as influenza, Klebsiella, or Pseudomonas.
  • the putative cloned MBL coding sequence will allow construction of an expression vector that will facilitate isolation of the MBL polypeptide sequence, which can be purified for nasal or oral delivery to subjects at risk of infection or infected with influenza or diarrhea caused by mannose or MBL-like adhesin containing pathogens.
  • the purified MBL could be delivered vaginally before or after intercourse to inactivate mannose or MBL-like adhesin containing pathogens associated with sexually transmitted disease.
  • Saliva was aseptically collected from healthy human subjects and plated on the Lactobacillus Rogosa (Difco) agar plates and incubated at 37°C for 24-48 h anaerobically. For liquid cultures, a single colony was transferred to Lactobacillus MRS broth incubated at 37°C for 16-24 h. When required, the culture was stored at -70°C with 15% glycerol.
  • Lactobacillus cells (10 7 /10 ⁇ l) from cultures of late log or stationary phase were washed with phosphate-buffered saline (PBS) and combined on a glass slide with Saccharomyces cerevisiae (10 6 /10 ⁇ l) grown in YPD broth at 37°C for 24 h and harvested similarly by resuspending in PBS.
  • the bacterium and yeast mixture was incubated for 10 min at room temperature. The cells were observed under a light microscope to detect the presence or absence of coaggregation of the bacteria and yeast cells.
  • Representative results obtained with Lactobacillus fermentum OLB- 19a are shown in Fig. 1.
  • Panel A shows OLB- 19a alone
  • panel B shows S.
  • panel C shows OLB- 19a and S. cerevisiae
  • panel D shows a OLB- 19a and S. cerevisiae in the presence of mannose (50 mM).
  • Lactobacillus fermentum OLB- 19a causes clumping of yeast cells (Fig. 1C), which does not occur in the presence of 50 mM mannose (Fig. ID).
  • the Gram-positive temperature-sensitive transposon-like plasmid pGh9:ISS7 was used to mutagenize the MBL + strain L. fermentum OLB- 19a.
  • the plasmid was transformed into OLB- 19a by electroporation (Wei et al, 1995).
  • the transformants were selected on MRS agar plates supplemented with 5 ⁇ g/ml erythromycin at 28°C for 48 - 72 h in a candle jar.
  • the transformants were tested for plasmid integration rate by replica-plating with one plate incubated at 42°C and the other at 28°C.
  • a transformant displayed a 1% plasmid integration rate at 42°C was selected for further studies. Mutagenesis was achieved by shifting a mid-exponential phase culture (10 ml) from 28°C to 42°C for 2 h without antibiotic selection, and subsequently plating on MRS agar plates supplemented with 5 ⁇ g/ml erythromycin and incubating at 37°C.
  • the red dye-stained yeast cells were extensively washed with PBS until the supernatant became clear. Mutant Lactobacillus clones were grown overnight in MRS broth in microtiter plates and transferred to nitrocellulose membranes. The bacterial cells were dried at room temperature and soaked in PBS. The red dye-stained yeast was used as a probe to react with the bacterial blot on the nitrocellulose membranes in a plastic pouch for 1 h on a rocker at room temperature. The membrane was extensively washed with PBS to remove unbound yeast. Among all tested mutants, only one, Tm-4A3, failed to bind the red dye- stained yeast. This mutant was used as a negative control isogenic MBL-negative strain for subsequent experiments.
  • the plasmid-tagged MBL gene was cloned in E. coli by isolating the mutant DNA, self-ligation, transformation of E. coli and selection of erythromycin resistant transformants at 28°C.
  • the DNA sequence of the cloned pGh9:ISS7 inserted gene is currently being determined.
  • Lactobacillus strains that co-aggregated with yeasts were tested for their binding to HIV envelope proteins.
  • the envelope protein (NIH AIDS Research and Reference Reagent Program, Cat# 386) of one representative strain, HIV-1 SF2 , was used for initial selection.
  • Overnight cultures of nine oral Lactobacillus strains and ten vaginal strains in MRS were harvested by centrifugation and washed twice with PBS and resuspended in PBS. The cells were subjected to brief sonication by a microsonic cell disrupter ( Komtes, Model KT50) to break clumps.
  • a microsonic cell disrupter Komtes, Model KT50
  • a 100 ⁇ l cell suspension (about 10 cells) was added, in triplates, to each well of microtiter plate pre-coated with 0.2 ⁇ g HIV- 1SF 2 gpl20.
  • the wells pre-coated with porcine gastric mucin were used as a negative control glycoprotein.
  • the isogenic MBL-negative mutant Tm-4A3 was used as a negative control Lactobacillus strain.
  • the microtiter plates were incubated for 20 min at room temperature. The unbound cells were removed by washing with PBS five times. The bound cells were eluted with 50 mM mannose and plated on MRS agar with serial dilutions for colony counts. Binding was also tested on protein coated glass slides.
  • MBL positive Lactobacillus strain OLB- 19a and its isogenic MBL- knockout mutant TM-4A3 were tested for the ability to bind to HIV envelope glycoproteins from various HIV or simian immunodeficiency virus (SIV) clades on microtiter plates, essentially as described in the previous section, or on glass slides that were first coated with protein, reacted with lactobaciUi, washed, and visualized by Gram staining.
  • SIV simian immunodeficiency virus
  • the viral envelope proteins were all obtained from NIH AIDS Research and Reference Reagent Program, including gpl20s of HIV CM235 (Cat# 2698), HIV Ba L (Cat# 4961), HIV S FI 6 2, (Cat# 7363) HIV SF 2 (Cat# 386), HIV.,TM (Cat# 4683), HIV C ⁇ ad eE (Cat# 3234), and HIV CN54 (Cat# 7749); gpl30 of SIV MaC239 (Cat# 2322); and gpl40 of SIV Mac i A ii (Cat# 2209).
  • HIV can be transmitted by cell-free or cell-associated viruses
  • HIV-infected cells such as the CD4 T- lymphocytes, in milk or semen, co-aggregate with bacteria, the cells will be trapped, thus reducing contact with HIV-target cells and cell-associated viral infection.
  • Three oral Lactobacillus strains (OLB- 19a, OLB-43b and Tm-4A3) were tested for co-aggregation with cell lines representing different HIV-target cells, including T lymphocytes (HPB-ALL-CD4, Jurket, CEMl, and CEM2), B lymphocytes (Jy), and monocytes (THP-J, THP-DC-SIGN, and U937), with and without 10 mM CaCl and/or 50 mM mannose.
  • the cells were cultured in RPMI-1640 medium (MediaTech, Cat# 10-040- CV).
  • OLB- 19a co-aggregated with all the HIV-target cells tested.
  • OLB-43b and the MBL mutant strain Tm-4A3 did not co-aggregate with any of these cells.
  • the co-aggregation between OLB— 19a and these HIV-target cells was blocked or reversed by 50 mM mannose or alpha-D-mannoside.
  • the cells were maintained on ice for about 1-2 h before mixing with HIV viruses.
  • Different concentrations of HIV-l ⁇ aL viruses (up to 2.1 x 10 /virion 50 ⁇ l) and Lactobacillus cells were mixed in the cell culture medium (without antibiotics) and incubated at 37°C for 1 h.
  • the bacterial cells were centrifuged for 5 min at 3000 xg and the pellet were assayed for bound HIV by the virus-capture p24 ELISA (Perkin Elmer, Cat# NEK050001KT).
  • the number of bound HIV was plotted as a function of the number of OLB- 19a cells (Fig. 5).
  • the number of virions bound is shown as a function of cell type (Fig. 6A), with OLB- 19a binding more than OLB-43b.
  • PBMC peripheral blood mononuclear cells
  • OLB- 19a cells were found to bind more HIV viruses, OLB-43b afforded greater protection of cells from infection with HIV (Fig. 6B). Increased HIV killing by OLB-43b may be due to secreted MBL and/or other potential viricidal acitivities, such as proteinase, acid and hydrogen peroxide.
  • Luciferase-tagged HIV can be used to monitor the rate of HIV infection because the infected cells will express the enzyme luciferase and will glow.
  • the 293T cells were co-transfected with the pHXB2 and pNL4-3.Luc.R-E- plasmids (catalog # 1069 and 3418; NIH AIDS Research and Reference Reagent Program) to produce luciferase-tagged HIV-l H ⁇ B2 viruses, which were used to infect U87 (U87.CD4.CXCR4: catalog# 4036) receptor cells after interacting with lactobaciUi. Briefly, 293T cells were seeded to 4x10 cells per 100 mm plate the day before transfection.
  • the virus was aspirated and replaced with U87 medium (Dulbecco's modification of Eagle's medium, Cat# 10-013-CV, MediaTech) and the cells were allowed to rest for another 24 h.
  • U87 medium Dulbecco's modification of Eagle's medium, Cat# 10-013-CV, MediaTech
  • the luciferase activity was measured in triplicate using the Luciferase Assay System from Promega (cat# E4030) and a Berthold FBI 2 luminometer running Sirius software.
  • the lactobaciUi were grown 37°C in MRS broth overnight to reach stationary phase.
  • the cells were washed with the receptor cell U87 medium and resuspended in 10% of its original volume (about 10 9 cells/ml).
  • OLB- 19a cells were subjected to a brief sonication to break up clumps.
  • the viruses (1 :16 dilution) and the MBL+ strains OLB- 19a, OLB-43b, or the MBL mutant strain Tm4A3 were mixed in equal volumes with the bacteria being diluted to varying concentrations (1 :2 through 1 :8) in the receptor cell media.
  • This medium contained 100 unit/ml penicillin G and 200 ⁇ g/ml streptomycin, which prevented growth of the lactobaciUi.
  • These samples of virus and bacteria were rocked gently at room temperature for 20 minutes and incubated on the bench for another 40 minutes. The samples were then centrifuged at 10,000 x g for 3 minutes and filtered through a 0.45 ⁇ m filter, added at 400 ⁇ l to the receptor cells, which was seeded the day before in a 12-well-plate to lxlO 4 cells per ml, and incubated at 37°C in a CO 2 incubator overnight. The following morning, new media was added to the cells.
  • luciferase activity (Luciferase assay kit E4030, Promega, Madison, WI) according to the manufacturer's instructions.
  • the luciferase readout was performed in triplicate at a tenfold dilution of receptor cell lysate on a triplicate set of infected receptor cells. The average of the luciferase triplicate readout was used as the output for each of the experiments and the triplicate of these experiments is used to calculate the standard deviation.
  • the results of inhibition of HIV infection by Lactobacillus strains are summarized in the bar graph shown in Fig. 7.
  • the Y axis shows the relative light units, which is correlated with HIV infection.
  • a comparison of a control infection (the first bar) is virus alone without the addition of bacteria..
  • OLB- 19a and mutant TM4A3 did not afford significant protection of the cells against the free virus.
  • OLB-43b afforded dose-dependent protection of cells against HIV even at the highest tested dilution (1:8).
  • the cells were maintained on ice for about 1-2 h before mixing with HIV or SIV viruses.
  • Different concentrations of HIV-2 or SIV viruses up to 2.1 x 10 8 /virion/50 ⁇ l
  • Lactobacillus cells were mixed in the cell culture medium (without antibiotics) and incubated at 37°C for 1 h.
  • the bacterial cells were centrifuged for 5 min at 3000 xg and the pellet were assayed for bound HIV-2 or SIV by the virus-capture p27 antigen ELISA using RETRO-TEK SIV (Zeptometrix, Catalog # 0801169).
  • a mock HIV model was constructed by cross-linking HIV-1 S F2 gpl20 to green fluorescent polystyrene beads (0.8 ⁇ m) (Spherotech, Inc., Libertyville, Illinois, USA) and mixed with isopropanol- killed, red fluorescence-stained L. fermentum OLB 19a cells (Live/Dead Stain, Molecular Probes, Eugene, Oregon, USA).
  • a fluorescent Olympus camera microscope was used to observe capture of mock HIV by red fluorescence stained lactobaciUi.
  • vaginal lactobacillus isolates were screened for co-aggregation of yeast as described above, and 26 isolates were found to bind yeast. Those 26 isolates were evaluated essentially as described above for the oral lactobacillus isolates. Twelve were found to adhere to gpl20-coated plates. These strains were subsequently tested for ability to block HIV-1 infections and to coaggregate with HIV-target cells, CD4+ ALL T lymphocytes and DC-SIGN+ monocytes as described above. Eight strains were found to block HIV-1 infection.
  • the five vaginal strains and six oral strains of Lactobacillus were further evaluated for the ability to protect against infection by bacterial vaginosis-associated bacteria (Esherichia coli ATCC14243, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Gardnerrella vaginalis ATCC14018, Gardnerrella vaginalis ATCC49145, and Mobiluncus curtisii subsp curtisii ATCC35241), or yeast associated with vaginal infections (Candida albicans ATCC 10231, C. krusei ATCC 14242, C. glabrata ATCC66032, C.
  • Lactobacillus OLB 19a The ability of Lactobacillus OLB 19a to inhibit fungal growth was further evaluated as follows.
  • the Lactobacillus OLB 19a culture was streaked in two parallel lines on a square MRS plate and incubated overnight at 37°C.
  • a MBL negative strain, Lactobacillus OLB21a was substituted for Lactobacillus OLB 19a in a parallel experiment.
  • Cultures of each of the six Candida isolates were spotted between the two Lactobacillus streaks, and at the far side of the plate, and incubated overnight. Inhibition of the yeast was observed by comparing the yeast drops in the two places on the same agar plate. The percentage inhibition was estimated by visually comparing yeast growth at the two sites at which the cultures were spotted.
  • Lyophilized preparations of select Lactobacillus strains were made as follows. Each bacterial strain was grown in 500 ml MRS broth to the stationary phase (about 16 h). The cells were harvested and washed once with phosphate-buffered saline, repelleted, and the pellet was resuspended in 5 ml 10% trehalose, sucrose, cellobiose, lactose or glucono-delton-lactone (GDL). The cell suspension was frozen at -80°C for 1 hour in a lyophilizing bottle. The freeze-drying was conducted with a Labconco Lyophilizer for about 20 h.

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Abstract

L'invention concerne des compositions contenant du lactobacille de lectine de fixation du mannose (MBL) positive, des procédés d'identification du lactobacille MBL positive, et des procédés d'utilisation correspondants.
PCT/US2005/007794 2004-03-10 2005-03-10 Nouvelles souches de lactobacille et procede d'utilisation correspondant WO2005086870A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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US9125768B2 (en) 2006-11-17 2015-09-08 Sca Hygiene Products Ab Hygiene tissue comprising a microbe-inhibiting composition
US9283297B2 (en) 2006-11-17 2016-03-15 Sca Hygiene Products Ab Sanitary article comprising a microbe-inhibiting composition
CN110680836A (zh) * 2018-06-19 2020-01-14 景岳生物科技(中国)有限公司 副干酪乳杆菌菌株gmnl-653用于制备改善狐臭组合物的用途
CN110804571A (zh) * 2019-12-02 2020-02-18 华中农业大学 一种复合乳酸菌制剂及在制备成饲料添加剂中的应用
EP4130258A1 (fr) * 2021-08-02 2023-02-08 Evonik Operations GmbH Micro-organismes affichant des récepteurs leurres viraux

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2087094A1 (fr) * 2006-11-17 2009-08-12 SCA Hygiene Products AB Lactobacillus fermentum ess-1, dsm17851 et son utilisation pour le traitement et/ou la prévention de la candidose et des infections des voies urinaires
JP2010509911A (ja) * 2006-11-17 2010-04-02 エスセーアー・ハイジーン・プロダクツ・アーベー カンジダ症及び尿路感染の予防及び／または治療のための発酵乳酸桿菌Ｅｓｓ−１、ＤＳＭ１７８５１及びそれの使用
EP2087094A4 (fr) * 2006-11-17 2010-10-06 Sca Hygiene Prod Ab Lactobacillus fermentum ess-1, dsm17851 et son utilisation pour le traitement et/ou la prévention de la candidose et des infections des voies urinaires
US8222020B2 (en) 2006-11-17 2012-07-17 Sca Hygiene Products Ab Biologically pure strain of Lactobacillus fermentum, strain Ess-1
US9125768B2 (en) 2006-11-17 2015-09-08 Sca Hygiene Products Ab Hygiene tissue comprising a microbe-inhibiting composition
US9283297B2 (en) 2006-11-17 2016-03-15 Sca Hygiene Products Ab Sanitary article comprising a microbe-inhibiting composition
CN110680836A (zh) * 2018-06-19 2020-01-14 景岳生物科技(中国)有限公司 副干酪乳杆菌菌株gmnl-653用于制备改善狐臭组合物的用途
CN110680836B (zh) * 2018-06-19 2021-04-02 景岳生物科技(中国)有限公司 副干酪乳杆菌菌株gmnl-653用于制备改善狐臭组合物的用途
CN110804571A (zh) * 2019-12-02 2020-02-18 华中农业大学 一种复合乳酸菌制剂及在制备成饲料添加剂中的应用
EP4130258A1 (fr) * 2021-08-02 2023-02-08 Evonik Operations GmbH Micro-organismes affichant des récepteurs leurres viraux
WO2023011828A1 (fr) * 2021-08-02 2023-02-09 Evonik Operations Gmbh Micro-organismes présentant des récepteurs leurres viraux

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