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WO2011070545A1 - Structure de type biofilm induite de manière virale et utilisations de celle-ci - Google Patents

Structure de type biofilm induite de manière virale et utilisations de celle-ci Download PDF

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WO2011070545A1
WO2011070545A1 PCT/IB2010/055747 IB2010055747W WO2011070545A1 WO 2011070545 A1 WO2011070545 A1 WO 2011070545A1 IB 2010055747 W IB2010055747 W IB 2010055747W WO 2011070545 A1 WO2011070545 A1 WO 2011070545A1
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cell
cells
viral
virus
biofilm
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Andres Alcover
Ana-Monica Pais Correia
Marie-Isabelle Bruna Thoulouze
Antoine Gessain
Martin Sachse
Stéphanie GUADAGNINI
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions

Definitions

  • the present invention relates to the field of virus transmission from cell to cell. More specifically, the present invention relates to the identification of a viral biofilm-Iike structure as a new mode of virus transmission and its use in methods of screening for a compound that affects formation and cohesion of such viral biofilm-Iike structure. The present invention also relates to compounds that affect virus transmission from cell to cell and use of such compounds in compositions and methods for the prevention and/or treatment of diseases or disorders associated with a virus inducing a biofilm-Iike structure.
  • HTLV Human T cell Lymphotropic Virus
  • HIV Human Immunodeficiency Virus
  • HAM/TSP HTLV-1 -associated myelopathy/tropical spastic paraperesis
  • HTLV-1 transmission requires cell contacts (Okochi, K., 1984; Donegan, E., 1994). It was shown that HTLV-1 -infected lymphocytes polarized their microtubules and viral components upon contact with other T cells, forming virological synapses where HTLV-1 could spread from cell to cell (Igakura, T., 2003; Barnard, A.L., 2005; Nejmeddine, M., 2005; Majorovits, 2008).
  • HIV-1 Human Immunodeficiency Virus
  • Figures 1 ,2 the formation of "polysynapses" by HIV-1 -infected T cells indicates that stable cell polarization is not absolutely required for HIV-1 transmission (Rudnicka, D., 2009).
  • dendritic cells can be infected by cell-free HTLV-1 and transmit the virus to T lymphocytes (Jones, K.S., 2008).
  • the present invention concerns a method of screening for a compound that affects formation or cohesion of viral biofilm-like structure, comprising the steps of:
  • the present invention also concerns a method of screening for a compound that affects virus transmission from cell to cell, comprising the steps of:
  • the present invention further concerns the method of the invention, wherein the viral biofilm-like structure consists in a structure comprising:
  • - carbohydrate rich matrix such as sialyl-Lewis X or sLeX, and/or
  • - adhesion molecules such as collagen or agrin and/or cellular linker proteins such as tetherin, galectin-3 or CD62.
  • the present invention further concerns the method of the invention, wherein the virus is a lymphotropic virus.
  • the present invention further concerns the method of the invention, wherein the virus is HTLV or HIV.
  • the present invention further concerns the method of the invention, wherein the lymphotropic virus consists of HTLV-1 , or HIV-1.
  • the present invention further concerns a compound that affects formation and/or cohesion of virus biofilm-like structure obtained by contacting a cell with a virus naturally involved in virus biofilm-like structure formation or cohesion.
  • the present invention further concerns a compound that affects virus transmission from cell to cell obtained by contacting a cell with a virus naturally involved in virus biofilm-like structure formation or cohesion.
  • the invention further provides a composition comprising the compound of the invention and a pharmaceutical acceptable carrier.
  • the present invention also concerns a method for preventing and/or treating diseases or disorders associated with a virus inducing a biofilm-like structure in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the composition of the invention.
  • the present invention also provides the use of a therapeutically effective amount of a composition of the invention for preventing and/or treating a subject against a biofilm-like structure forming associated disease.
  • the subject is a human.
  • the subject is immunocompromised.
  • the biofilm-like structure forming associated disease is caused by a lymphotropic virus.
  • the lymphotropic virus is HTLV or HIV.
  • the virus is HTLV-1 or HIV-1.
  • Figure 1 HTLV-1 cell to cell transmission; Intracellular localization of Gag p 9 complexes in HTLV-1 -infected T-cell conjugates.
  • Naturally infected T-cells CD4-
  • CD4- Naturally infected T-cells purified from the PBMCs of HTLV-1 -seropositive donors. It shows the superposition of staining with anti-p-tubulin mAb (dark grey) and anti-Gag p19 mAb (light grey).
  • (1a) shows unpolarized Gag protein in isolated T-cell.
  • HTLV-1- infected CD4+ and normal CD4+ T cell Gag p19 (dark grey).
  • HTLV-1 -infected T cells were marked with carboxyfluorescein succinimidyl ester (CFSE) (light grey).
  • CFSE carboxyfluorescein succinimidyl ester
  • the transmission picture is superimposed on a 0.4- ⁇ confocal fluorescence single section. Scale bar, 5 ⁇ .
  • CD4+-CD8+ T cell conjugates were allowed to form for 40 min.
  • FIG. 2 HTLV-1 cell to cell transmission; a scheme of HTLV-1 cell to cell transmission showing a virological synapse, as commonly described on the basis of the seminal work by Igakura (Igakura, T., 2003).
  • FIG. 3 HTLV-1 cell to cell transmission -
  • Figure 4 The infectious viral particles encased in an extracellular matrix "cocoon", enable their efficient and protected transfer between cells. Extracellular matrix components and linker proteins ensure the cohesiveness and adhesiveness of these infectious structures. Disrupting these structures by competition would expose encased viral particles to the existing immune response and thus, facilitate the clearance of the virus.
  • Figure 5 The viral biofilm-like structure ensures the protected transfer of infectious viral particles from one cell to another one either through tight junctions or through more distant contacts, such as filopodia or nanotubes, recently shown to support transfer of retroviruses between cells (Sherer, 2007; Sowinski, 2008).
  • FIG. 6 The viral biofilm-like structure ensures the rapid and efficient transfer of infectious viral particles from one cell to another one. Scanning electron microscopy of chronically infected CD4+ T cells forming multiples contacts with target cells (Jurkat CD4+ T cells). Conjugates were allowed to form for 5 min. Secondary electron image detection (SEI) allows visualization of the morphology of cells as well as biofilm like structures already adhered to the surface of target cells.
  • SEI Secondary electron image detection
  • Figure 7 The viral biofilm-like structure ensures the rapid and efficient transfer of infectious viral particles from one cell to another one. Another scanning electron microscopy of chronically infected CD4+ T cells forming multiples contacts with target cells (Jurkat CD4+ T cells). Conjugates were allowed to form for 5 min. Secondary electron image detection (SEI) allows visualization of the morphology of cells as well as biofilm like structures already adhered to the surface of target cells.
  • SEI Secondary electron image detection
  • FIG. 8 Extracellular HTLV-1 assemblies are carbohydrate-rich structures. Confocal microscopy of CD4+ T cells from HAM/TSP individuals showing cell surface glycoproteins stained with Lens culinaris (LCA), and viral proteins stained with a HAM/TSP serum and with Gag-p19 antibody. Projections of three- dimensional reconstructions are shown. Bottom panels show the volume of co- localization of LCA and serum staining (green + red means LCA + TSPser). Examples of infected (a,b) and non-infected cells (c) are shown. Representative of three experiments were carried out with cells from two different patients.
  • LCA Lens culinaris
  • FIG. 9 Extracellular matrix and linker proteins are enriched in HTLV-1 viral assemblies. Confocal microscopy of CD4 + T cells from HAM/TSP individual immunostained for sialylLewisX (sLeX) (grey, column B), for the extracellular matrix components depicted within panels and for viral proteins (Gag-p19 antibody or HAM-TSP serum) (grey, column A). Infected (left) and uninfected cells (middle) are shown. Projections of three-dimensional reconstructions are shown. Right panels show quantification of the absence (-), the presence (+), or the accumulation (++) of each matrix component in cells from several HAM/TSP subjects (depicted by #) and in C91/PL and MT2 cell lines. Data represent means ⁇ S.D. of two independent observer's counts. Representative of three experiments for (a), two for (b) and (d) and five for (c) and (e).
  • Figure 10 Composition of viral biofilms - Expression of SLeX, Collagen, Galectin are induced by the virus; Expression of Agrin and Tetherin is induced by the infection.
  • FIG. 11 HTLV-1 extracellular viral assemblies are carbohydrate-rich structures.
  • Table 10 Listed plant lectins were used to stain cell surface glycoproteins of CD4+ T cells from two HAM/TSP individuals, C91/PL and MT2 cell lines. The inventors ranked the general intensity of lectin cell staining (+++; ++; +; -), as well as the presence and accumulation (Acc.) of lectin labeling in viral assemblies .
  • CD4+ T lymphocytes from a healthy donor were transfected with HTLV-1 molecular clone (pCMV-HT1).
  • Cells were surface stained with LCA (column B) and for viral proteins using HAM/TSP serum (column A) and Gag-p19 mAb (column A, arrow).
  • a projection of three-dimensional image reconstructions and the volume of colocalization are shown (green + blue means LCA + Gag-p19 mAb).
  • CD4+ T cells from HAM/TSP individuals were surface stained with ConA. Total fluorescence intensity in infected and non infected cells was quantified. Mann- Whitney test, (p ⁇ 0.0001 ) was used for statistical analysis. A representative experiment is shown out of two experiments carried out.
  • Figure 12 Clusters of viral components are on the surface of infected cells.
  • SEI Secondary electron image detection
  • YAG backscattered detection
  • Figure 13 HTLV-1 viral components cluster at the cell surface and in the uropod.
  • (a-c) Confocal microscopy of CD4+ T lymphocytes from an asymptomatic HTLV-1 carrier (a), C91/PL (b) and MT2 (c) cell lines showing staining of surface glycoproteins with ConA (grey) and HTLV-1 proteins with HAM/TSP serum (thin arrows) and Gag-p19 mAb (thick arrows). Medial confocal sections (Mid. Sect.), or projections of three dimensional reconstructions (3D proj.) are shown.
  • (d,e) CD4+ T lymphocytes from HAM/TSP individuals stained with the uropod markers ICAM-1 (d) and P-ERM antibodies (e), and Gag-p19 (d), (e) antibody (arrows).
  • FIG 14 Scanning electron microscopy of extracellular viral assemblies.
  • C91/PL (a), MT2 (b) and Jurkat cells (Jkt) (c) were stained by immunogold with Env-gp46 antibody.
  • SEI detector allows the visualization of cell morphology and YAG detector the visualization of gold particles.
  • Jurkat cells were used as negative control (c).
  • Figure 15 Extracellular viral assemblies on the surface of HTLV-1 -infected cells.
  • Black arrowheads show mature virus particles (dense core surrounded by an envelope labeled by gold particles). The white arrowhead shows "empty vesicles" lacking dense cores and gold labeling.
  • Figure 16 Selective accumulation of extracellular matrix proteines in HTLV-1 viral assemblies,
  • (a-c) Confocal microscopy of CD4+ T cells from HAM/TSP subjects showing staining of fibronectin, a-dystroglycan (ct-dystr.), or neuropilin 1 (Nrp-1 ) and viral proteins using HAM/TSP serum or Gag-p19 antibody.
  • (d,e) MT2 and C91/PL showing staining of agrin and Gag-p19. Projections of three-dimensional reconstructions are shown in a-c and medial optical sections in d,e.
  • FIG. 17 Treatment with Heparinase III or metalloproteinases fractionates HTLV-1 extracellular viral assemblies. Confocal microscopy showing CD4+ T cells from HAM/TSP individuals (a), and C91/PL (left) and MT2 (right) cell lines treated with heparinase III or with a metalloproteinase cocktail and stained for surface glycoproteins with ConA, and for viral proteins with HAM/TSP serum and Gag-p19 antibody. Representative of three experiments carried out.
  • Figure 18 HTLV-1 extracellular viral assemblies spread at cell contacts, (a) Confocal microscopy of CD4 + T cell conjugates from HAM/TSP individuals showing cell surface glycoproteins stained with Con A (grey), and viral proteins stained with HAM/TSP serum (very light grey) and with Gag-p19 antibody (arrows). Conjugates between infected and uninfected cells were identified by the presence of cortical serum + Gag-p19 labeling, and stronger Con A staining of infected cells (i), compared to noninfected cells (ni) (see also Figure 20). Merge images of XY-medial optical sections, XY-projection of a three-dimensional reconstruction and XZ-medial optical section are shown.
  • Figure 19 Confocal microscopy of CD4 + T cell conjugates from HAM/TSP individuals showing cell surface glycoproteins stained with Con A (grey), and viral proteins stained with HAM/TSP serum (arrows) and with Gag-p19 antibody (arrows). Conjugates between infected and uninfected cells were identified by the presence of cortical serum + Gag-p19 labeling, and stronger Con A staining of infected cells, compared to noninfected cells. Extracellular viral assemblies accumulate not only at the junction of the cells but also outside the contact zone.
  • Figure 20 Cell-to-cell transfer of extracellular viral assemblies
  • (a-c) Confocal microscopy of CD4+ T cells from HAM/TSP individuals showing staining of surface glycoproteins with ConA and viral proteins with HAM/TSP serum and Gag-p19 antibody. The confocal microscope was set to observe Gag-p19 fluorescence in the cell cortex. Extracellular viral assemblies are therefore saturated.
  • Infected cells in conjugates display cortical Gag-p19 staining, as well as higher ConA staining compared with non-infected cells from the same individual,
  • Figure 21 Transfer of extracellular HTLV-1 assemblies at cell contacts. Transmission electron microscopy showing a MT2-Jurkat cell conjugate stained for Env-gp46 by immunogold.
  • FIG. 22 Relevance of extracellular HTLV-1 assemblies for cell-to-cell transmission. Viral assemblies can be removed by cell washing. C91/PL cells, or primary CD4 + T cells from HAM/TSP patients were left unwashed, or washed by extensive pipetting, dilution and centrifugation in the absence (washed), or in the presence of heparin (washed + heparin), (a) Schematic representation of the experiment, (b) FACS on cells; Flow cytometry measurements of Gag-p19 that remained associated to C91/PL.
  • Figure 23 Cell washes do not perturb conjugate formation. Phase-contrast microscopy of C91/PL infected cells left unwashed (left), or washed mechanically, by pipetting, dilution and centrifugation, in the absence (middle), or in the presence (right) of heparin. After washing out the heparin, infected cells were co-cultured with the luciferase reporter cell line at a 1 :2 ratio similarly to the experiment in Figure 22. A representative experiment is shown out of two experiments carried out.
  • Figure 24 Function of the viral biofilm in viral transmission. Infectious viral particles (a) are embedded in an extracellular matrix cocoon adhering to the cell surface that can be detached by specific enzymatic treatments, such as heparinase III (10 U/ml) (b).
  • Figure 25 Scheme summarizing a major pathway for HTLV-1 cell-to cell transmission.
  • Extracellular adhesive structures are composed of infectious particles embedded in an extracellular matrix, forming protective carbohydrate-rich structures which rapidly adhere and infect other lymphocytes.
  • FIG. 26 Comparison of viral biofilm-like structure (HTLV-1) (a) and bacterial biofilm (Bordetella bronchiseptica), (b) (Parise, 2007) both composed of infectious microbes embedded in an extracellular matrix, forming a structure that stocks, concentrates, protects and disseminates a microorganism.
  • HTLV-1 viral biofilm-like structure
  • bacterial biofilm Bacilla bronchiseptica
  • FIG. 27 Polarized budding of HIV-1 (T lymphocytes )- T cells were also apparently induced to form microvilli by adhering to plastic, as prominent microvilli were observed at the base of the cells.
  • the most interesting aspect of adhesion to plastic was polar secretion of HIV. This phenomenon was striking when cells were viewed with the SEM. In many cells, hundreds of virions were seen in the area immediately adjacent to the plastic, among microvilli, and on the plastic adjacent to the cell. However, virions were virtually absent from the rest of the cell surface (a) SEM of a MOLT/HIV-1
  • Figure 28 Polarized budding of HIV-1 (T lymphocytes ); TEM through the base of a MOLT4/HIV-1 iMB cell 40 min after being placed onto plastic in serum-free medium.
  • the matrix material (m) indicates that this section has transected the cell immediately above the plastic. Numerous mature and budding virions (arrows) are observed in association with the plasmalemma. Magnification, x 18,000. (Pearce-Pratt, 1994)
  • Figure 29 TEM of a MOLT4/HIV-1 nn B cell 40 min after the addition of colchicine. Virions are observed exclusively on the tip of the pseudopod (arrows). Magnification, x8,000.(Pearce-Pratt, 1994)
  • Figure 30 Polarized budding of HIV-1 (monocytes)
  • Figure 31 Use according to claim 10, wherein the biofilm-like structure forming associated disease is caused by Clusters of viral particles were localized on the surface of HIV-1 -infected cells. Confocal microscopy of HIV-1 infected primary CD4 + T cells showing cell surface glycoproteins stained with Con A and viral proteins stained with Gag-p17 antibody. Medial optical sections are shown. Gag- pi? is the process form of the viral protein Gag only present in mature viral particles.
  • FIG 32 Extracellular HIV-1 assemblies are carbohydrate-rich structures. Confocal microscopy of HIV-1 -infected CD4 + T cells showing cell surface glycoproteins stained with Glycine max lectin, and viral proteins stained with Gag-p17 antibody. Projections of three-dimensional reconstructions are shown.
  • Figure 33 HIV-1 extracellular viral assemblies spread at cell contacts. Confocal microscopy of HIV-1 infected CD4 + T cell conjugated with non- infected T cell showing cell surface glycoproteins stained with Con A, and viral proteins stained with Gag antibody. Merge images of XY-medial optical sections and a XY-projection of a three-dimensional reconstruction are shown. Arrows point to Gag + clusters at the cell surface incubated for 30 min, then stained with Gag antibody.
  • virus cell-to-cell transmission (such as HTLV-1 or HIV-1 cell-to-cell transmission) does not necessarily occur directly through synaptic contacts. Rather, HTLV-1 virions bud at the plasma membrane and are transiently kept in adhesive extracellular structures rich in carbohydrates and composed of virally-induced extracellular matrix and linker proteins whose synthesis and/or rearrangement are virally induced. These are reminiscent of bacterial biofilms (Stewart, P.S. 2008). These extracellular viral assemblies rapidly adhere during cell contacts to other lymphocytes supporting their infection.
  • viral biofilm-like structure refers to a virally induced extracellular structure comprising viral particles, extracellular matrix, linker proteins and/or adhesion molecules with carbohydrate moieties.
  • a method of screening for a compound that affects formation of viral biofilm-like structure comprises a step of contacting a cell with a virus naturally involved in viral biofilm-like structure and at least one candidate compound, and a step of identifying the compound that affects the formation of the viral biofilm-like structure or the cohesion of such biofilm.
  • the expression "formation of virus biofilm-like structure” refers to either already formed biofilm or to a virus infected cell which is in conditions that induces or facilitates the formation of a viral biofilm.
  • the invention is thus concerned with a compound that affects formation and cohesion of viral biofilm-like structure obtained by the above detailed method.
  • adhesion molecules may consist for instance of collagen, agrin or cellular linker proteins such as tetherin, galectin-3 and CD62.
  • a method of screening for a compound that affects virus transmission from cell to cell comprises a step of infecting a cell with a virus naturally involved in viral biofilm-like structure for a time sufficient to form a virus biofilm-like structure, a step of contacting the virus infected cell with a non-infected cell and at least one candidate compound, and a step of identifying the compound that affects transmission of said virus from the infected cell to the non-infected cell.
  • the invention is also concerned with a compound that affects virus transmission from cell to cell obtained by the previously mentioned method.
  • contemplated compound of the invention may for instance interfere with the adherence and/or the cohesion of the viral biofilm to other cells upon cell contact. More particularly, the contemplated compound may inhibit :
  • the term "cell” refers to any cell that is capable of being infected by a virus which naturally forms a viral biofilm-like structure.
  • a cell may be a lymphocyte.
  • virus used in accordance with the present invention may be for instance a lymphotropic virus, such as HTLV or HIV, and not limited to HTLV-1 or HIV-1.
  • the compounds obtained by the screening/selecting methods of the invention may be used in many ways in the prevention and/or treatment of diseases or disorders associated with viruses inducing biofilm-like structure.
  • another embodiment of the present invention relates to a composition for preventing and/or treating such diseases or disorders, and more particularly for preventing and/or impairing the formation and/or the cohesion of virally-induced biofilm-like structures.
  • the composition of the present invention advantageously comprises a compound of the invention and an acceptable carrier.
  • the term “impairing” refers to a process by which the formation or development of a virally-induced biofilm-like structure is affected or completely destroyed.
  • the term “preventing” when referring to biofilm formation refers to a process by which the formation, the cohesion, or the development of a virally-induced biofilm-like structure is obstructed or delayed.
  • preventing refers to a process by which the symptoms of such diseases or disorders is obstructed or delayed.
  • treating it is intended, for the purposes of this invention, that the symptoms of such diseases or disorders be ameliorated or completely eliminated.
  • an acceptable carrier means a vehicle for containing the compounds obtained by the method of the invention that can be administered to a subject without adverse effects.
  • Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions.
  • Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i. e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
  • composition of the invention may also comprise other anti-viral agents well known in the art.
  • the amount of compounds obtained by the method for preventing and/or treating diseases or disorders associated with viruses inducing biofilm-like structure according to the invention is preferably a therapeutically effective amount.
  • a therapeutically effective amount of compound obtained by the screening/selecting method of the invention is the amount necessary to allow the same to perform their inhibitor role in accordance with the present invention without causing overly negative effects in the host to which the composition is administered.
  • the exact amount and/or dosage of compounds obtained by the method of the invention to be used and the composition to be administered will vary according to factors such as the mode of administration, the patient's weight, age, or condition, as well as the other ingredients in the composition.
  • the composition of the invention may be given to a subject through various routes of administration.
  • the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions.
  • sterile injectable preparations such as sterile injectable aqueous or oleaginous suspensions.
  • suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os.
  • Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age, the condition, and the weight of the subject to be treated. Any other methods well known in the art may be used for administering the composition of the invention particularly to avoid contamination via biologic fluids such as milk or sperm.
  • another embodiment of the invention is to provide a method for preventing and/or treating diseases or disorders associated with a virus inducing a biofilm-like structure in a subject, the method comprising the step of administering to the subject (e.g. a human which may be immunocompromised) a therapeutically effective amount of a composition as defined above.
  • the subject e.g. a human which may be immunocompromised
  • HTLV-1 Human T cell leukemia virus type 1
  • HTLV-1 -infected T lymphocytes form "virological synapses", but the mechanism of HTLV-1 transmission remains poorly understood.
  • the inventors show here that HTLV- 1 -infected T lymphocytes transiently store viral particles in extracellular platforms that remain adhered to the cell surface. The viral particles are protected being poorly accessible to antibodies and proteases in the extracellular milieu. These carbohydrate rich extracellular assemblies are held together and attached to the cell surface by virally induced extracellular matrix components, including collagen and/or agrin, and/or cellular linker proteins, like tetherin, galectin-3 and/or CD 62.
  • Extracellular viral assemblies rapidly adhere to other cells upon cell contacts allowing virus spread and infection of target cells. Their removal strongly reduces the ability of HTLV-1 -producing cells to infect target cells.
  • the present results unveil a novel virus transmission mechanism based on the generation of extracellular viral particle assemblies whose structure, composition and function resemble those of bacterial biofilms.
  • HTLV-1 biofilm-like structures represent a major route for virus transmission from cell to cell.
  • HTLV-1 -infected cell lines MT2 and C91/PL, and HTLV-1 Env gp46 (0.5a) mAb were obtained from the NIH AIDS Research and Reference Reagent Program.
  • Jurkat cells clone J77cl20 was previously described (Thoulouze, M.I., 2006).
  • Rabbit Ab to Agrin (C95) was a gift from M. Riiegg (University of Fribourg, Switzerland).
  • Mouse Env gp46 4D4 antibody (Grange, M.P., (1998)) was a gift of C. Pique (Institut Pasteur, Paris).
  • Mouse mAb to Gag-p19 (TP-7) was obtained from Zeptometrix.
  • HTLV-1 Blot Kit Serum from a HAM/TSP individual (#1378) with strong reactivity against a panel of viral proteins (HTLV-1 Blot Kit, Genelabs Diagnostics) was used (AG, unpublished).
  • HTLV-1 protein labeling was not found neither in cells from healthy donors, nor in non-infected T cell lines, nor in a percentage of cells from HTLV-1 -infected patients, supporting the specificity of the inventor's antibodies to HTLV-1 proteins ( Figures 8, 9, 10).
  • CD1 5s (CSLEX1) and ICAM- 1 (LB2) mAbs were obtained from Becton-Dickinson. Rabbit polyclonal Ab to human collagen l/l l/l I l/l V/V was obtained from AbD Serotec.
  • Mouse mAb to galectin-3 was obtained from Affinity BioReagents. Goat Abs to BST-2 (tetherin) (K- 15 and N-17) were obtained from Santa Cruz Biotechnology. Cyanine 3 (Cy3)-coupled Abs to mouse lgG2b, goat IgG, human Ig, mouse Ig and fluorescein-coupled mouse lgG2a, were obtained from Jackson Immunoresearch. Fluorescein-coupled mouse lgG1 was obtained from Southern Biotechnology. Alexa 488 -coupled Ab to fluorescein was obtained from Molecular Probes. Colloidal gold protein A was obtained from the University Medical Center Utrecht, The Netherlands.
  • Phycoerythrin-coupled Ab to mouse IgG was obtained from Beckman-Coulter. Metaloproteinases MMP Multipack- 1 , was obtained from Biomol. Heparinase III was obtained from Sigma. Plant lectins ( Figure 11), ConA, LCA, UEAI, SB A, PNA, WGA and PHA-E were obtained from Sigma and HHL was obtained from Vector Laboratories.
  • the inventors purified peripheral blood mononuclear cells through Ficoll-Hypaque centrifugation and isolated CD4 + T cells by negative selection, using magnetic cell sorting (Miltenyi Biotec, MACS).
  • the inventors placed the CD4 + -enriched T cell population in culture for 18 hours as previously described (Igakura, T., 2003; Nejmeddine, M., 2005).
  • the inventors analyzed cells from seventeen HAM/TSP subjects and two asymptomatic carriers that contained 2-20 % of HTLV-1 -infected cells per sample, as assessed by immunofluorescence against viral proteins after 18 h of ex vivo culture.
  • Heparinase and MMP treatments were treated with heparinase III (10 U/ml) (Hep. Ill), or with a cocktail of metalloproteinases (10 nM) (MMP) for 1 h at 37°C in serum-free medium.
  • Immunofluorescence, confocal microscopy analysis and quantification of fluorescence intensity The inventors performed them as previously described (Thoulouze, M.I.,, (2006)). Cells were fixed with 4 % paraformadehyde for 30 min at room temperature. Surface staining (e.g., lectin staining) was performed in the absence of detergent. Intracellular cellular proteins and Gag-p19 were stained incubating fixed cells in solutions containing 0.05 % saponin. Before staining, the inventors blocked nonspecific protein binding by incubating the coverslips for 15 min in 5% FCS 0.05% saponin in PBS.
  • the inventors treated cells after paraformadehyde fixation with 100 % methanol, 40 min at -20°C.
  • the inventors carried out confocal microscopy analysis on a Zeiss LSM5 10 using a x 63 objective. Z-series of optical sections at 0.2 ⁇ increments were acquired.
  • the inventors treated the images by deconvolution using Huygens software in order to reduce the fluorescence noise of the images. Three- dimensional image reconstructions of cells and the calculation of the volume of co-localization were done with Imaris software.
  • Samples were dehydrated through a graded series of 25, 50, 75 and 95 % ethanol solution for 5 min each time and 10 min in 100 % ethanol followed by critical point drying with C0 2 in a CPD Baltec apparatus.
  • the dried specimens were mounted on stubs with carbon tape and ion sputtered with 15 nm carbon layer.
  • Analysis of Secondary Electron Image (SEI) and backscattered images (YAG detector) was performed on a Jeol JSM 6700F microscope with a field emission gun operating at 5 kV.
  • SEI Secondary Electron Image
  • YAG detector backscattered images
  • MT2 cells were mixed with Jurkat cells at 1 :1 ratio for different times and cell suspensions ( 0 6 cells/cover slip) were put onto poly-L-lysine-coated cover slips, let to sediment for 3 min, centrifuged at 47 x g for 1 min and fixed with 2% formaldehyde in 0.1 M phosphate buffer pH 7.4 for 5 min and than for 1 h with 4 % formaldehyde in the same buffer. After fixation, remaining free aldehydes were quenched for 10 min in 50 mM NH CI, Cells were then labeled using saturating concentrations of the human anti-Env gp46 (0.5 mA) antibody and protein A coupled to 10 nm gold.
  • the inventors isolated peripheral blood CD4 + T cells from healthy donors as described above, and then transfected the pCMV- XMT plasmid (gift of F. Delbecque (Institut Pasteur, Paris) using Amaxa according to the manufacturer's instructions. The inventors analyzed the transfected cells by fluorescence microscopy after 24 hrs of culture.
  • the inventors left HTLV-1- infected cells unwashed, or washed three times in RPMI-1640 serum free medium and incubated 1 h at 37°C in the absence or in the presence of 50 pg.ml "1 heparin (Sigma-Aldrich) in serum-free medium. After washing three times in RPMI-1640 10 % FCS, the inventors processed cells for immunofluorescence analysis and flow cytometry, or to infect luciferase reporter gene target cells. The inventors detected viral antigens on cell culture supernatants by Gag-p19 ELISA according to the manufacturer's instructions (Zeptometrix).
  • Luciferase reporter gene assay The inventors stably transfected Jurkat cells with a plasmid containing the luciferase gene under the control of HTLV-1 LTR (gift of R. Mahieux (Institut Pasteur, Paris)). The inventors co-cultured Luciferase reporter cells, in a 96-cell plate round bottom, with HTLV-1 -infected cells at a cell ratio of 2:1 , respectively. After 24 hours, the inventors assessed luciferase activity using a Promega luciferase kit assay and a TR717 Microplate Luminometer (Berthold Technologies). When indicated, reporter cells were incubated for 48 h with the reverse transcriptase inhibitor AZT at 50 ⁇ prior to coculture with HTLV-1 -infected cells. AZT was left in the co- culture.
  • Viral components cluster on the surface of infected cells
  • HTLV-1 -infected lymphocytes were shown to cluster Gag and Env viral proteins at the cell cortex, whereas Tax was localized at the microtubule- organizing center (MTOC) and in the nucleus (Igakura, T., 2003; Nejmeddine, M., 2005; Mazurov, D., 2006).
  • MTOC microtubule- organizing center
  • infected cells polarized their MTOC, and Gag, Env and Tax proteins towards the cell contact. Then, viral components appeared in non infected cells, indicating that transfer of virus to target cells had occurred.
  • the inventors therefore carried out confocal and electron microscopy analyses in primary CD4 + T cells from HTLV-1 -infected individuals, and in the chronically infected cell lines C91 /PL and MT2.
  • Gag-p19 was shown to cluster in discrete areas of the cell cortex (Igakura,, T., 2003; Nejmeddine, M., 2005; Mazurov, D,, 2006) that could be intracellular compartments where viral particles assembled and/or accumulated ready to be transferred to target cells.
  • the inventors used sera from HAM/TSP individuals reactive against various viral proteins, and Gag-p19-specific monoclonal antibody (mAb).
  • the inventors then labeled cell surface glycoproteins with the lectin Concanavalin-A (ConA). To obtain maximal image definition, deconvolution of confocal images and three- dimensional reconstruction were performed.
  • Viral protein clusters correspond to extracellular viral assemblies
  • the inventors therefore performed transmission electron microscopy (Figure 15).
  • the inventors observed on the cell surface of MT2 cells mature viral particle assemblies, displaying electron-dense cores and Env gold- labeling (Figure 15b, black arrowheads), as well as empty vesicles ( Figure 15b, white arrowhead).
  • the inventors observed a mesh of electron-dense material among the viral particles, putatively, extracellular matrix ( Figure 15b, arrow).
  • double immunogold staining on thawed cryo-sections of MT2 cell pellets revealed extracellular assemblies of mature viral particles containing both Env and Gag-p19 ( Figure 15c, arrowheads).
  • Extracellular viral assemblies are carbohydrate-rich structures
  • Extracellular matrix components provide attachments for viruses, including HTLV-1 , facilitating their entry (Jones, K.S., 2005; Pinon, J.D., 2003). They could also facilitate the attachment and concentration of budding viral particles into extracellular viral assemblies.
  • the inventors used plant lectins recognizing glycans enriched in the extracellular matrix (Neu, T.R., 1999; McClure, S.F., 1997 ( Figure 11, table).
  • the inventors took advantage of the fact that HTLV-1 gp46 is poorly glycosylated if compared with glycoproteins of many other viruses, including HIV- 1 gp 120 (Le Blanc, I., 2001 ; Coffin, J.M, 1997).
  • sLeX sialyl-LewisX
  • extracellular viral particles are embedded in a carbohydrate- rich structure that is induced and spatially reorganized by viral infection.
  • HTLV-1 assemblies contain extracellular matrix and linker proteins
  • Agrin a heparan sulfate proteoglycan that cross-links cell surface receptors and is involved in neural, immunological and viral synapses (Dityatev, A., (2006); Khan, A.A., (2001 ); Alfsen, A., (2005)), was also concentrated in viral clusters ( Figure 9c, Figure 16d-f).
  • a-dystroglycan a heparan sulfate proteoglycan that clusters with agrin in synaptic structures (Zhang, J., (2006)), did not accumulate (Figure 16b), indicating preferential concentration of some proteoglycans in these structures.
  • neuropilin a transmembrane protein involved in HTLV-1 entry, present in HTLV-1 virological synapses (Ghez, D., 2006) and in immunological synapses (Tordjman, R. , 2002), was not concentrated in extracellular viral assemblies (Figure 16c).
  • Galectin-1 and galectin-3 are secreted by T lymphocytes (Rabinovich, G.A., (2009)), and upregulated by HTLV-1 infection (Hsu, D.K., 1996; Gauthier, S., 2008).
  • the inventors found galectin-3 ( Figure 9d), but not galectin- 1 , clustered in viral assemblies at the surface of cells from HAIWTSP individuals. However, in MT2 and C9 1 /PL cells, galectin-3 was poorly expressed and did not cluster with viral particles.
  • Tetherin (BST-2/CD3 17), is an interferon-inducible transmembrane protein whose degradation by the HIV-1 Vpu protein provokes an extracellular clustering of HIV-1 virions reminiscent of the extracellular HTLV-1 assemblies shown here (Neil, S.J., 2008; Van Damme, N., 2008). Since, HTLV-1 lacks vpu-like sequences, tetherin may facilitate virion attachment to the cell surface. Consistently, it was shown that Vpu expression enhanced HTLV-1 release (Jouvenet, N., 2009).
  • Virological synapses were described as organized cell contacts allowing direct virus transfer through synaptic clefts (Igakura, T., 2003; Majorovits, E., 2008). Consistently, the inventors observed that HTLV-1 -infected T cells formed tight cell conjugates and transferred viral proteins to non-infected cells. However, three-dimensional views and xz-sections of cell conjugates surface-stained with ConA, showed extracellular viral assemblies overlapping cell contacts and bridging the gap between both cell surfaces, rather than filling contact sites (Figure 18a, right panel).
  • Extracellular viral assemblies forming the biofilm not only accumulate at the junction of the two cells, but also outside this contact zone, bridging the gap between both cell surfaces (Figure 19).
  • Infected cells were distinguished from non-infected cells by the presence of cortical viral proteins and higher ConA labeling (Figure 20a-c).
  • Conjugates between CD4 + T cells from HAM/TSP individuals and healthy subjects also displayed viral assemblies on the surface of infected and target cells and on the sides of cell contacts ( Figure 18b). Extracellular matrix components were transfer together with viral components to target cells ( Figure 20d-f). Similar results were found in MT2-Jurkat cell conjugates. At 10 min, «35 % of conjugates showed Gag-p19 labeling on target cells, increasing until 1 h, ( Figure 18c, d arrowheads). The large majority of cell conjugates displayed viral assemblies on the sides rather than in the center of the synapse ( Figure 18c,e arrows).
  • extracellular HTLV-1 assemblies can be rapidly transferred outside the synaptic zone to the surface of other lymphocytes.
  • extracellular viral assemblies account for over 80 % of the infectious capacity of HTLV-1 infected cells.
  • HTLV-1 infected cells produce and transiently store virions in extracellular adhesive structures rich in extracellular matrix components and linker proteins that are critical for HTLV-1 cell-to-cell transmission (Figure 25).
  • the present results are in line with previously reported data showing extracellular clusters of HTLV-1 viral particles associated to electron dense material (Goussen, A., 1990; Poiesz, B.J., 1980; Zacharopoulos, V.R., 1992).
  • Extracellular HTLV-1 assemblies are strikingly pronounced of bacterial biofilms, which are composed of bacteria held together by a carbohydrate-rich extracellular matrix that ensures cohesion, protection, adhesiveness to a substrate and spreads upon fragmentation (Stewart, P.S., 2008) ( Figure 26, Parise et al 2007).
  • Bacterial biofilms are rich in exopolysaccharides produced by bacteria (Stewart, P.S., 2008) but extracellular matrix proteins like fibrinogen, or lectins like galectin-3, produced by host cells can also cooperate with bacterial proteins enhancing cohesion and adhesiveness (Fowler, M., (2006); Moran, A.P., 2008; Gupta, S.K., 1997; Bonifait, L., 2008).
  • HTLV-1 hijacks similar host cell proteins enhancing their expression, modifying their carbohydrate composition, or their spatial organization to build extracellular assemblies.
  • HTLV-1 may utilize host cell adhesion molecules for attachment to the cell surface and for clustering at the uropod.
  • concentrating virions in these structures may locally increase "infectious titers" and help to convey the virus from cell to cell.
  • extracellular composition and structure of extracellular matrix, including collagen and galectin-3 is modified by the ⁇ -irradiation of cell cultures (El Nabout, R., 1989; Joo, H.G., 2001 ). Therefore, irradiation could reshape the structure of extracellular viral assemblies enhancing viral transmission, explaining why irradiation of HTLV-1 producing cells increases their efficiency to infect other cells in vitro.
  • Extracellular viral assemblies were resistant to strong shear flow during extensive pipetting, suggesting that they can defy physiological fluid dynamics in vivo.
  • irregular surfaces help the formation and stability of bacterial biofilms.
  • some bacteria induce host cell surface reorganization to resist shear flow (Mikaty, G., 2009).
  • HTLV-1 - infected cells generate numerous ruffles and filopodia that could maintain viral assemblies adhered to the surface of virus producing cells, but facilitate their transfer to other cells during cell contacts.
  • viral assemblies might be rapidly transferred to other T lymphocytes or dendritic cells during cell contacts in secondary lymphoid organs.
  • Dendritic cells may get infected and/or transfer again HTLV-1 assemblies to T lymphocytes (Jones, K.S., 2008).
  • dendritic cells could also process viral assemblies and present viral antigens to T lymphocytes, triggering and maintaining the anti-HTLV-1 immune response.
  • EXAMPLE 2 Biofilm-like extracellular viral assemblies mediate HIV-1 cell-to- cell transmission at virological synapses
  • the inventors also performed experiments with HIV-1 infected lymphocytes in order to observe whether they form extracellular viral assemblies at their cell surface ( Figures 27, 28, 29 30).
  • the rabbit anti-HIV-1 Gag Ab was a gift from the NIH AIDS Research and Reference Reagent Program.
  • Affinity purified anti-human CD62L mouse mAb to CD62L (clone DREG-56) was obtained from eBioscience.
  • the other antibodies, lectins or reagents, have been described in Example 1.
  • J.CaM1.6 (JCaM) a Lck-deficient line derived from Jurkat, was previously described (Straus, D. B., and A. Weiss. 1992).
  • Peripheral blood mononuclear cells were purified through Ficoll-Hypaque centrifugation and CD4 + T cells were isolated by negative selection, using magnetic cell sorting (Miltenyi Biotec, MACS).
  • Jurkat (parental or Lck-deficient) cell lines or primary CD4+T cells isolated from healthy donors were infected with HIV-1 X4-tropic HIV-1 strain NL4.3 (HIVwt) as previously described (Thoulouze et al. 2006).
  • the inventors analyzed HIV-1 - infected cells, as assessed by immunofluorescence against viral proteins after 72 h of infection.
  • the inventors performed them as previously described (Thoulouze, M.I., 2006). Cells were fixed with 4 % paraformadehyde for 30 min at room temperature. Surface staining (e.g., lectin staining) was performed in the absence of detergent. Intracellular cellular proteins and Gag-p19 were stained incubating fixed cells in solutions containing 0.05 % saponin. Before staining, the inventors blocked nonspecific protein binding by incubating the coverslips for 15 min in 5% FCS 0.05% saponin in PBS. For collagen staining, the inventors treated cells after paraformadehyde fixation with 100 % methanol, 40 min at -20°C.
  • the inventors carried out confocal microscopy analysis on a Zeiss LSM5 10 using a x 63 objective. Z-series of optical sections at 0.2 pm increments were acquired. The inventors treated the images by deconvolution using Huygens software in order to reduce the fluorescence noise of the images. Three-dimensional image reconstructions of cells and the calculation of the volume of co-localization were done with I maris software.
  • the inventors carried out confocal microscopy analyses in primary CD4 + T cells infected with HIV-1.
  • Gag was shown to cluster in discrete areas of the cell cortex that could be intracellular compartments where viral particles assembled and/or accumulated ready to be transferred to target cells.
  • the inventors used Gag-specific antibodies, and the inventors labeled cell surface glycoproteins with the lectin Concanavalin-A (ConA) on in vitro infected primary CD4+ T cells from healthy donors, or on cells from the Jurkat T cell line.
  • ConA lectin Concanavalin-A
  • Extracellular viral assemblies are carbohydrate-rich structures
  • the inventors used a panel of plant lectins recognizing glycans enriched in the extracellular matrix.
  • the inventors observed that, like ConA ( Figure 31), most lectins evenly stained the surface of uninfected cells. However, some lectins, such as Glycine Max, stained much more strongly extracellular viral assemblies ( Figure 32). The colocalization between lectin labeling and viral markers was however weak, indicating that lectins did not primarily bind viral Env glycoprotein, but glycoconjugates enriched in extracellular viral assemblies.
  • extracellular viral particles are embedded in a carbohydrate- rich structure, likely made of extracellular matrix, that is induced and spatially reorganized by viral infection.
  • HIV-1 assemblies contain linker proteins
  • CD62L is an adhesion molecule whose surface expression and further cleavage and release are regulated by T cell activation.
  • T cell activation controls the formation of HIV-1 extracellular viral assemblies
  • the inventors have observed in preliminary experiments that the size of HIV-1 extracellular clusters depend on a key T cell activation molecule, the protein tyrosine kinase Lck. Thus in T cells lacking this kinase (JCaM cells), the extracellular viral clusters are larger. Clusters became smaller again when the inventors corrected the levels of expression of this kinase by gene transfection.
  • HIV-1 may control the formation of extracellular viral assemblies by controlling the activation machinery of infected cells.
  • HIV-1 -infected T lymphocytes form extracellular viral assemblies at their cell surface, which are reminiscent to those that the inventors had observed on the surface of HTLV-1 -infected lymphocytes.
  • HIV-1 and HTLV-1 extracellular assemblies share some characteristics, like the presence of carbohydrate-rich structures and linker proteins, like CD62L. They seem however distinct in some respects, such as their size and the presence of particular carbohydrate moieties.
  • HIV-1- infected blood mononuclear cells form an integrin- and agrin-dependent viral synapse to induce efficient HIV-1 transcytosis across epithelial cell monolayer.
  • Galectin-3 binds to Helicobacter pylori O-antigen: it is upregulated and rapidly secreted by gastric epithelial cells in response to H. pylori adhesion. Cell Microbiol 8, 44-54 (2006)
  • Tordjman, R. et al. A neuronal receptor, neuropilin- 1 , is essential for the initiation of the primary immune response. Nat Immunol 3, 477-82 (2002) Van Damme, N. et al. The Interferon-lnduced Protein BST-2 Restricts HIV- 1 Release and Is Downregulated from the Cell Surface by the Viral Vpu Protein. Cell Host Microbe (2008)

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Abstract

La présente invention se rapporte au domaine de la transmission virale de cellule à cellule. Plus spécifiquement, la présente invention porte sur l'identification d'une structure virale de type biofilm en tant que nouveau mode de transmission virale et sur son utilisation dans des procédés de balayage pour un composé affectant la formation et la cohésion d'une telle structure virale de type biofilm. La présente invention porte également sur des composés affectant la transmission virale de cellule à cellule et sur l'utilisation de tels composés dans des compositions et des procédés de prévention et/ou de traitement de maladies ou de troubles associés à un virus induisant une structure de type biofilm.
PCT/IB2010/055747 2009-12-10 2010-12-10 Structure de type biofilm induite de manière virale et utilisations de celle-ci Ceased WO2011070545A1 (fr)

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WO2004019970A2 (fr) * 2002-08-30 2004-03-11 K.U. Leuven Research And Development Derives antibiotiques de glycopeptides
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