CA2688105A1 - Virally induced biofilm-like structure and uses thereof - Google Patents

Abstract

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-like structure as a new mode of virus transmission and its use in methods of screening for a compound that affects formation of such viral biofilm-like structure or for a compound that affects virus transmission and cohesion 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-like structure.

Description

VIRALLY INDUCED BIOFILM-LIKE STRUCTURE AND USES THEREOF
FIELD OF THE INVENTION

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-like structure as a new mode of virus transmission and its use in methods of screening for a compound that affects formation of such viral biofilm-like structure or for a compound that affects virus transmission and cohesion 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-like structure.

BRIEF DESCRIPTION OF THE PRIOR ART

HTLV- 1 infects 15-20 millions people worldwide. Although most of the infected individuals are asymptomatic, 5-10 % develop T cell leukemia, or inflammatory syndromes such as HTLV- 1 -associated myelopathy/tropical spastic paraperesis (HAM/TSP) 1.

HTLV- 1 transmission requires cell contacts 2,3. 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 4-7. Likewise, virological synapses mediate efficient cell-to-cell transmission of human immunodeficiency virus (HIV- 1) 8'9. Hence, HTLV- 1 and HIV- 1 appear to hijack the T cell polarization machinery to direct virus budding to cell contacts, favoring virus transfer to target cells 4,7,9,10 Nonetheless, the formation of "polysynapses" by HIV- 1 -infected T cells indicates that stable cell polarization is not absolutely required for HIV- 1 transmission Unlike T cells, dendritic cells can be infected by cell-free HTLV- 1 and transmit the virus to T lymphocytes 12 SUMMARY
The present invention concerns a method of screening for a compound that affects formation of viral biofilm-like structure, comprising the steps of:

- contacting a cell with a virus naturally involved in viral biofilm-like structure and at least one candidate compound; and - identifying the compound that affects the formation of said viral biofilm-like structure or the cohesion of said biofilm.

The present invention also concerns a method of screening for a compound that affects virus transmission from cell to cell, comprising the steps of.-- infecting a cell with a virus naturally involved in viral biofilm-like structure for a time sufficient to form a viral biofilm-like structure;
- contacting said virus infected cell with a non-infected cell and at least one candidate compound; and - identifying the compound that affects transmission of said virus from the infected cell to the non-infected cell.

The present invention further concerns a compound obtained by the methods of the invention and a composition comprising said compound 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 a composition as defined above.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Clusters of viral components are on the surface of infected cells.
(a,b) Confocal microscopy of primary CD4+ T cells from HAM/TSP individuals showing cell surface glycoproteins stained with Con A (green) and viral proteins stained with HAM/TSP serum (red) and with Gag-p19 antibody (blue).
Medial optical sections (Mid. Sect.), or projections of three-dimensional reconstructions are shown. (3D proj.). (c,d) Scanning electron microscopy of primary CD4+ T cells from HAM/TSP individuals showing Env-gp46 stained by immunogold (15 nm). Secondary electron image detection (SEI) allows visualization of cell morphology, whereas backscattered detection (YAG) allows gold particle detection. Arrows point the areas enlarged in lower panels. Drawings schematize the shape of the cell and the area displaying putative Env+ viral clusters. (a,b) Cells displaying on their surface one, or several, viral protein clusters (arrows). (c,d) Env+ virus clusters on the cell surface, or on the edge of the membrane lamellipodium that adheres the cell to the coverslip (arrows). Representative of ten experiments carried out for a and b and three for c and d with cells from different subjects.

Figure 2: Extracellular viral assemblies on the surface of HTLV- 1 -infected cells. (a,b) Transmission electron micro scopy of MT2 cells on coverslips displaying Env-gp46 stained by immunogold before Epon embedding. (b) Enlargement of the area framed in a. Insets show areas framed in b and marked 1, 2. 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. The arrow points to the electron dense mesh, putatively extracellular matrix. (c) Cryo-sections of MT2 cell pellets showing immunogold staining of Env-gp46 (15 nm gold) and Gag-p19 (10 nm gold) (P = plasma membrane). (d-f) CD4+ T cells from HAM/TSP individuals showing Gag-p19 costaining by immunofluorescence and immunogold (10 nm). Infected cells identified by fluorescence microscopy (d) were processed for transmission electron microscopy (e, f). The framed area in e is enlarged in f. Arrowheads show mature virus particles (dense core surrounded by an envelope labeled by gold particles). Representative of five experiments performed for a and b, and three for c and d-f.

Figure 3: 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, green), and viral proteins stained with a HAM/TSP serum (red) and with Gag-p19 antibody (blue). Projections of three-dimensional reconstructions are shown.
Bottom panels show the volume of co-localization of LCA and serum staining.
Examples of infected (a,b) and non-infected cells (c) are shown.
Representative of three experiments carried out with cells from two different patients.

5 Figure 4: Extracellular matrix and linker proteins are enriched in HTLV- 1 viral assemblies. Confocal microscopy of CD4+ T cells from HAM/TSP individuals immunostained for sialylLewisX (sLeX) (red), for the extracellular matrix components depicted within panels and for viral proteins (Gag-p19 antibody or HAM-TSP serum) (green). 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 5: 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 (green), and viral proteins stained with HAM/TSP serum (red) and with Gag-p19 antibody (blue). 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) (Supplementary Fig. 6). Merge images of XY-medial optical sections, XY-projection of a three-dimensional reconstruction and XZ-medial optical section are shown. (b) CD4+ T lymphocytes from HAM/TSP individuals co-cultured for 1 h with healthy donor CD4+ T lymphocytes labeled with DDAO-SE dye (blue) 9, stained with ConA (green) and with Gag-p19 antibody (red).
Projections of three-dimensional reconstructions are shown. Arrows point to Gag+ clusters at the cell surface and at the side of the contact. (c) MT2 cells cocultured with CFSE-labeled Jurkat cells (Jkt, green) incubated for 10 min, or 1 hr, then stained with Gag-p19 mAb. A medial optical section is shown.
Gag+ clusters on the surface of the infected cells (arrows) and of target cells (arrowheads) are shown. (d) Percentage of Jurkat target cells with Gag+
clusters versus time of co-culture. (e) Percentage of cells conjugates displaying Gag clustered on the side or in the middle of the synapse versus time of co-culture. (f) T lymphocytes from HAM/TSP individuals immunogold labeled for Env gp46. (g) MT2 cells in contact Jurkat cells (Jkt) for 5 min. Right sub-panels are higher magnification of the framed areas. SEI and YAG detection reveals, respectively, cell morphology and gold particles. Representative of ten experiments performed for a, three for b, c, f, five for g and two for d, e.
Figure 6: Relevance of extracellular HTLV- 1 assemblies for cell-to-cell transmission. 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) Flow cytometry measurements of Gag-p19 that remained associated to C91/PL. c:
ELISA measurements of Gag-p19 released into the supernatant of C9 1 /PL
cells after the different wash treatments. (d-g) Luciferase activity of reporter Jurkat cells co-cultered for 24 h, with the following: (d) C9 1 /PL cells that underwent the different wash treatments; (e) C9 1/PL cells in the presence or absence of AZT; (t) 100x-concentrated supernatants from C91/PL cells that underwent the different wash treatments; (g) HAM/TSP primary CD4+ T cells that underwent the different wash treatments. Data represent means S.D. of triplicates. Representative experiment out of four experiments for b-d, three for e and f, and two for g.

Supplementary Figure 1: 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 (green) and HTLV-1 proteins with HAM/TSP serum (red) and Gag-p19 mAb (blue). 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) (red), and Gag-p19 antibody (green) (arrows).

Supplementary Figure 2: Scanning electron microscopy of extracellular viral assemblies. C91/PL (a), MT2 (b) and Jurkat cells (Jkt) 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).

Supplementary Figure 3: HTLV-1 extracellular viral assemblies are carbohydrate-rich structures. (Table) 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 . (a) CD4 + T cells from a HAM/TSP individual surface stained with PNA and for viral proteins using HAM/TSP serum (red) and Gag-p19 mAb (blue). (b) CD4+ T lymphocytes from a healthy donor were transfected with HTLV-1 molecular clone (pCMV-HT1). Cells were surface stained with LCA (green) and for viral proteins using HAM/TSP serum (red) and Gag-p19 mAb (blue). A
projection of three-dimensional image reconstructions and the volume of colocalization are shown. (c) 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.

Supplementary Figure 4: 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 (a-dystr.), or neuropilin 1 (Nrp-1) (green) and viral proteins using HAM/TSP serum or Gag-p19 antibody (red). (d,e) MT2 and C91/PL showing staining of agrin (green) and Gag-p19 (red). Projections of three-dimensional reconstructions are shown in a-c and medial optical sections in d,e. (f) Scanning electron microscopy of C91/PL
cells showing immunogold labeling of agrin (15 nm). Arrows indicate zones corresponding to enlarged areas. A representative experiment is shown out of three experiments carried out for a-e and two for f.

Supplementary Figure 5: 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 (a, red) and Gag-p19 antibody (a, blue, b-d, red). Representative of three experiments carried out.

Supplementary Figure 6: 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 (green) and viral proteins with HAM/TSP serum (red) and Gag-p19 antibody (blue). 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. (d-f) CD4+ T cells from HAM/TSP individuals stained for tetherin, agrin or collagen and viral proteins with Gag-p19 antibodies. A
representative experiment is shown out of five experiments carried out for a-c and two for V.

Supplementary Figure 7: Transfer of extracellular HTLV-1 assemblies at cell contacts. Transmission electron microscopy showing an MT2-Jurkat cell conjugate stained for Env-gp46 by immunogold. (a) Low magnification of a cell conjugate displaying extracellular viral assemblies spreading from the surface of the MT2 to the Jurkat (Jkt) cell at the side of the cell contact (white arrow). (b,c) High magnification of the center (b) and the lateral area (c) of the cell contact marked by frames in a. Mature viral particles displaying dense cores and Env-gold labelling are pointed by arrowheads. The black arrow points to the mesh of electron dense material, putatively extracellular matrix. A representative experiment is shown out of five experiments carried out.

Supplementary Figure 8: 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 Fig. 6. A representative experiment is shown out of two experiments carried out.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that virus cell-to-cell transmission (such as HTLV-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 13. These extracellular viral assemblies rapidly adhere during cell contacts to other lymphocytes supporting their infection. The present findings uncover a novel and major mode of virus cell transmission which may serve as a model to develop new anti-viral agents 5 to prevent or treat diseases or disorders associated with viruses capable of forming a viral biofilm-like structure.

As used herein, the expression "viral biofilm-like structure" refers to a virally induced extracellular structure comprising viral particles, extracellular matrix, linker proteins and adhesion molecules with carbohydrate moieties.

1. Methods of screening/selection and compounds According to an aspect of the invention, there is provided a method of screening for a compound that affects formation of viral biofilm-like structure. The method 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.

As used herein, 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 induce or facilitate the formation of a viral biofilm.

According to a related aspect, the invention is thus concerned with a compound that affects formation and cohesion of viral biofilm-like structure obtained by the above detailed method.

One of the art will understand that the above mentioned contemplated compound of the invention may for instance inhibit the formation of the carbohydrate rich matrix, or interfere with the clustering of the molecules in the viral biofilm-like structure. Such adhesion molecules may consist for instance of collagen, agrin and cellular linker proteins such as tetherin, galectine-3 and CD62.

According to another aspect of the invention, there is provided a method of screening for a compound that affects virus transmission from cell to cell.
The method 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.

According to a related aspect, the invention is also concerned with a compound that affects virus transmission from cell to cell obtained by the previously mentioned method.

One of the art will understand that such a contemplated compound of the invention may for instance interfere with the adherence and the cohesion of the viral biofilm to other cells upon cell contact. More particularly, the contemplated compound may inhibit:

^ the binding of the molecule (e.g. linker protein)forming the biofilm to the surface to cell and to the surface to target cell ^ The binding and the interaction of the molecules of the biofilm among them.

It will be understood that, in accordance with the present invention, the term "cell" refers to any cell that is capable of being infected by a virus which is naturally forms a viral biofilm-like structure. For instance, such a cell may be a lymphocyte.

It will be further understood that the virus used in accordance with the present invention may be for instance a lymphotropic virus, such as, but not limited to HTLV, HIV.

2. Methods of use and compositions 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.

In this connection, 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.

As used herein, 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. As used herein, the term "preventing" when referring to biofilm formation, refers to a process by which the formation or development of a virally-induced biofilm-like structure is obstructed or delayed.

When referring in general to diseases or disorders associated with a virus inducing a biofilm-like structure, the term "preventing or prevention" refers to a process by which the symptoms of such diseases or disorders is obstructed or delayed. By the term "treating" is intended, for the purposes of this invention, that the symptoms of such diseases or disorders be ameliorated or completely eliminated.

As used herein, the expression "an acceptable carrier" means a vehicle for containing the compounds obtained by the method of the invention that can be administered to a animal host 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.

Further agents can be added to the composition of the invention. For instance, the composition of the invention may also comprise other anti-viral agents well known in the art.

The amount of compounds obtained by the method of 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 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, 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. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These 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 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.

Yet, 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 present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLE: Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses Introduction Human T cell leukemia virus type 1 (HTLV- 1) is a lymphotropic retrovirus whose cell to cell transmission requires cell contacts. 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 as carbohydrate rich extracellular assemblies that are held together and attached to the cell surface by virally induced extracellular matrix components, including collagen and agrin, and cellular linker proteins, like tetherin, galectin-3 and CD 62.

5 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 10 function resemble those of bacterial biofilms. HTLV- 1 biofilm-like structures represent a major route for virus transmission from cell to cell.

METHODS
Cells lines and reagents. HTLV-1-infected cell lines MT2 and C91/PL, and 15 HTLV-1 Env gp46 (0.5a) mAb were from the NIH AIDS Research and Reference Reagent Program. Jurkat cells clone J77c120 was previously described 48. Rabbit Ab to Agrin (C95) was a gift from M. Ruegg. Mouse Env gp46 4D4 antibody 49 was a gift of C. Pique. Mouse mAb to Gag-p19 (TP-7) was from Zeptometrix. 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). We did not find HTLV-1 protein labeling in cells from healthy donors, in non-infected T cell lines, and in a percentage of cells from HTLV- 1 -infected patients, supporting the specificity of our antibodies to HTLV- 1 proteins (Fig. 3, 4). CD1 5s (CSLEXI) and ICAM-1 (LB2) mAbs were from Becton-Dickinson. Rabbit polyclonal Ab to human collagen I/ll/III/IV/V was from AbD Serotec. Mouse mAb to galectin-3 (clone A3A1 2) was from Affinity BioReagents. Goat Abs to BST-2 (tetherin) (K- 15 and N-17) were from Santa Cruz Biotechnology. Cyanine 3 (Cy3)-coupled Abs to mouse IgG2b, goat IgG, human Ig, mouse Ig and fluorescein-coupled mouse IgG2a, were from Jackson Immunoresearch. Fluorescein-coupled mouse IgG1 was from Southern Biotechnology. Alexa488-coupled Ab to fluorescein was from Molecular Probes.

Colloidal gold protein A was from the University Medical Center Utrecht, The Netherlands. Phycoerythrin-coupled Ab to mouse IgG was from Beckman-Coulter. Metaloproteinases MMP Multipack- 1, was from Biomol. Heparinase III was from Sigma. Plant lectins (Supplementary Fig. 3), ConA, LCA, UEAI, SBA, PNA, WGA and PHA-E were from Sigma and HHL was from Vector Laboratories. HTLV-1 expression plasmid containing the wild-type proviral clone XMT, allowing the expression of the full-length HTLV- 1 genome under the control of the cytomegalovirus immediate early promoter 50 was kind gift from Dr Frederic Dellebecque.

Isolation of primary CD4+ T lymphocytes. 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 4,6. 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. Cells were treated with heparinase III (10 U/ml) (Hep. III), 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 48 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 Imaris software.

Scanning electron microscopy. Cell suspensions containing 106 cells/slide were put onto poly-L-lysine-coated coverslips, let sediment for 3 min, centrifuged at 47 x g for 1 min and fixed for 20 min at room temperature in 4 % paraformaldehyde. Samples were washed in phosphate buffered saline (PBS) and incubated for 10 min with 50 mM NH4CI in PBS. Samples were then washed in PBS 1 % BSA and incubated for 1 h with the anti-HTLV-1 Env gp46 (0.5 mA) in PBS 1 % BSA. After washing in PBS 0.1 % BSA and in PBS 1 % BSA, samples were incubated for 15 min with protein A coupled to 15 nm or 20 nm gold particles, washed in PBS and fixed in 2.5 %
glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 overnight at 4 C. Cells were washed 3 times in 0.2 M cacodylate buffer (pH 7.2) for 5 min each, post-fixed for 1 h in 1 % (wt/vol) osmium tetroxide in 0.2 M cacodylate buffer, and then rinsed with distilled water. 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 CO2 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.

Transmission electron microscopy. Before contact with HTLV- 1 chronically infected MT2 cell suspensions, Jurkat cells were incubated for 1 h with BSA coupled to 6 nm gold (Aurion, The Netherlands) with a final OD520 of 2. Non-endocytosed BSA-gold was washed away with warm medium containing 10% FCS. MT2 cells were mixed with Jurkat cells at 1:1 ratio for different times and cell suspensions (106 cells/cover slip) were put onto poly-L-lysine-coated cover slips, let sediment for 3 min, centrifuged at 47 x g for 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 NH4CI, 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 samples were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 before post-fixation with OsO4. During dehydration in a graded series of EtOH samples were stained with 1.2% uranyl acetate in 70% EtOH and after dehydration embedded in Epon and polymerized at 60 C for 48h. After polymerization blocks were sectioned with a Leica ultramicrotome. After staining with uranyl acetate and lead citrate samples were observed at 80 kV with a Jeol 1010 microscope and images were acquired with a CCD camera (Keenview, Softlmaging). For immunolocalization on thawed cryo sections cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Processing of cells for ultrathin cryosectioning and double immunolabeling according to the protein A-gold method was done as described by Slot et al. 51 Correlative immunofluorescence and transmission electron microscopy.
Primary CD4+ T cells from HAM/TSP patients cell suspensions, prepared as described above, were sediment on MatTek glass bottom dishes, containing gridded cover slips to localize the cells of interest. Cells were fixed with 2%
formaldehyde and 0.1% glutaraldehyde in 0.2 M Hepes buffer, pH 7.4 for 5 min, washed with 4% formaldehyde and fixation was continued for 30 min with 4%

formaldehyde in 0.2 M Hepes, pH 7.4. After quenching of free aldehyde groups with NH4CI (50 mM), cells were permeabilized with 0.05 % saponin in PBS and labeled with mouse anti-Gag p19 antibody, followed by goat anti-mouse coupled to Alexa-488 and 10 nm gold (Invitrogen). After acquisition of images in a fluorescence microscope (Leica SP5), samples were fixed with 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 and embedded in EPON as described above. After polymerization the cell of interested was localized and the area containing the cell was prepared for sectioning with a Leica ultramicrotome. After staining with uranyl acetate and lead citrate all samples were observed at 80 kV with a Jeol 1010 microscope and images were acquired with a CCD camera (Keenview, Softimaging).

Cell transfection. The inventors isolated peripheral blood CD4+ T cells from healthy donors as described above, and then transfected the pCMV-XMT
(gift of F. Delbecque) plasmid using Amaxa according to manufacturer. The inventors analyzed transfected cells by fluorescence microscopy after 24 hrs of culture.

Detachment of extracellular virus assemblies from infected cells. 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). The inventors co-cultured Luciferase reporter cells, in a 96-5 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 pM prior to coculture with 10 HTLV-1-infected cells. AZT was left in the co-culture.

RESULTS
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-15 organizing center (MTOC) and in the nucleus 4,6,14 Upon contact with other T
cells, infected cells polarized their MTOC, and Gag, Env and Tax proteins towards the cell contact. Then, viral components appeared in noninfected cells, indicating that transfer of virus to target cells had occurred. This supported the notion of virological synapses as being virus-induced organized cell contacts 20 through which HTLV- 1 was transmitted from cell to cell 4,6,7. However, the precise localization of viral components and the subcellular compartments involved remained ill defined.

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 4,6,14 that could be intracellular compartments where viral particles assembled and/or accumulated ready to be transferred to target cells. To localize viral proteins with respect to the plasma membrane the inventors used sera from HAM/TSP individuals reactive against various viral proteins (data not shown), and Gag-p19-specific monoclonal antibody (mAb), and the inventors 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.

Strikingly, cortical clusters of viral proteins were on the cell surface, appearing as single or multiple clusters, faintly labeled by ConA (Fig. la,b).
Similar results were found in samples from all the seventeen HAM/TSP
subjects and two asymptomatic carriers studied (Supplementary Fig. 1a and data not shown). The inventors did not detect viral protein clusters inside infected cells, although their plasma membrane appeared evenly and dimly stained by HAM/TSP sera and by Gag-p19 antibodies (Fig. 1b). Notably, ConA staining was significantly higher in HTLV- 1 positive cells (Supplementary Fig. 3c). Finally, MT2 or C91 /PL cells also displayed viral protein clusters at the cell surface (Supplementary Fig. 1 b,c). In polarized cells, viral proteins were often found on the uropod, as assessed by the enrichment in ICAM-1 and phosphorylated ERM (ezrin-radixin-moesin) (Supplementary Fig. 1 d,e), suggesting that adhesion and cytoskeletal structures may maintain viral components polarized on the cell surface.

Viral protein clusters correspond to extracellular viral assemblies By scanning electron microscopy and immunogold staining of Env glycoprotein (gp46), cells from HAM/TSP individuals displayed putative Env+
viral particle clusters embedded in a smooth material (Fig. 1 c,d). Virus clusters were of different sizes and could be observed at the edge of membrane extensions (Fig. 1d). Similar Env+ virus clusters were observed in C91/PL
and MT2, but were absent from uninfected lymphocytes (Supplementary Fig.
2a-c). These data suggested that extracellular clusters of Env+ viral particles were at the surface of infected cells. However, the inventors could not rule out that Env glycoprotein shed from viral particles could concentrate in heparan-sulfate-proteoglycan-enriched areas 15. The inventors therefore performed transmission electron microscopy (Fig 2). The inventors observed on the cell surface of MT2 cells mature viral particle assemblies, displaying electron-dense cores and Env gold-labeling (Fig 2b, black arrowheads), as well as empty vesicles (Fig 2b, white arrowhead).
Moreover, the inventors observed a mesh of electron-dense material among the viral particles, putatively, extracellular matrix (Fig 2b, arrow).
Moreover, double immunogold staining on thawed cryo-sections of MT2 cell pellets revealed extracellular assemblies of mature viral particles containing both Env and Gag-p19 (Fig. 2c, arrowheads). Finally, cells from HAM/TSP
individuals were observed using correlative immunofluorescence and transmission electron microscopy (Fig. 2d-f). Positive cells by immunofluorescence (Fig. 2d) displayed extracellular Gag-p19 immunogold-labeled mature viral particles with membrane and dense core (Fig. 2f, arrowheads).

Altogether, the light and electron microscopy observations show the presence of extracellular viral assemblies on the surface of HTLV- 1 -infected T
lymphocytes.

Extracellular viral assemblies are carbohydrate-rich structures Extracellular matrix components provide attachments for viruses, including HTLV-1, facilitating their entry 15,16. They could also facilitate the attachment and concentration of budding viral particles into extracellular viral assemblies.
To investigate this, the inventors used plant lectins recognizing glycans enriched in the extracellular matrix 17,18 (Supplementary Fig. 3, 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 19,20. The inventors observed that, like ConA (Fig. 1), most lectins evenly stained the surface of uninfected cells (Supplementary Fig. 3, table and data not shown). However, Lens culinaris (LCA) and Arachis hypogeae (PNA) lectins, although stained evenly uninfected cells, they much strongly stained extracellular viral assemblies (Fig. 3). The colocalization between lectin labeling and viral markers was however weak, indicating that lectins do not primarily bind viral Env glycoprotein, but glycoconjugates enriched in extracellular viral assemblies (Fig. 3a,b and Supplementary Fig. 3a). Likewise, uninfected primary T cells transfected with expression vectors encoding the full length HTLV- 1 genome exhibited LCA labeling on viral protein clusters (Supplementary Fig. 3b). Finally, the inventors observed that sialyl-LewisX
(sLeX), a tetrasaccharide involved in lymphocyte adhesiveness, overexpressed upon HTLV-1 infection 21,22 , concentrated in extracellular viral assemblies (Fig.
4a).

Therefore, 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 The inventors next searched for extracellular matrix components in HTLV-1 assemblies. Both collagen and fibronectin are overexpressed in HTLV-1 infected cells in a Tax-mediated manner 23,24. The inventors found collagen enriched in viral assemblies, but randomly distributed on uninfected cells (Fig. 4b). In contrast, neither fibronectin (Supplementary Fig. 4a), nor laminin (data not shown) were coclustered with virions. Agrin, a heparan sulfate proteoglycan that cross-links cell surface receptors and is involved in neural, immunological and viral synapses 25,26,27, was also concentrated in viral clusters (Fig. 4c, Supplementary Fig. 4d-f). Interestingly, a-dystroglycan, a heparan sufate proteoglycan that clusters with agrin in synaptic structures 28, did not accumulate (Supplementary Fig. 4b), indicating preferential concentration of some proteoglycans in these structures. Finally, neuropilin, a transmembrane protein involved in HTLV- 1 entry, present in HTLV- 1 virological synapses 29 and in immunological synapses 30, was not concentrated in extracellular viral assemblies (Supplementary Fig. 4c). Finally, treatment of cells with heparinases, or metalloproteinases, that hydrolyze heparan sulfate proteoglycans and various extracellular matrix proteins, respectively, lead to the detachment and fractionation of HTLV-1 assemblies (Supplementary Fig. 5 a-d). This further supports the involvement of extracellular matrix in the generation of these structures.

The inventors next hypothesized that galectins, (3-galactoside-binding lectins that form lattices that condition extracellular matrix properties, could also be involved. Galectin-1 and galectin-3 are secreted by T lymphocytes 31, and upregulated by HTLV-1 infection 32,33. The inventors found galectin-3, but not galectin-1, clustered in viral assemblies at the surface of cells from HAM/TSP
individuals (Fig. 4d, and data not shown). However, in MT2 and C9 1 /PL cells, galectin-3 was poorly expressed and did not clustered with viral particles (data not shown).

Finally, the inventors found tetherin concentrated in extracellular viral assemblies (Fig. 4e). 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 34,35Since, 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 36 Altogether, these data strongly support the involvement of extracellular matrix and linker proteins in the cohesion and attachment of HTLV-1 assemblies to the surface of infected cells.

Cell-to-cell transmission of extracellular viral assemblies Virological synapses were described as organized cell contacts allowing direct virus transfer through synaptic clefts 4,7. Consistently, the inventors 5 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 (Fig. 5a, right panel).
10 Infected cells were distinguished from non-infected cells by the presence of cortical viral proteins and higher ConA labeling (Supplementary Fig. 6a-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 (Fig. 5b). Extracellular matrix 15 components were transfer together with viral components to target cells (Supplementary Fig. 6d-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 1h, (Fig. 5c,d arrowheads). The large majority of cell conjugates displayed viral assemblies on the sides rather than in the 20 center of the synapse (Fig. 5c,e arrows).

Scanning electron microscopy showed Env+ virus clusters very close to cell contact sites (Fig. 5 f). At 5 min of contact, the inventors already observed Env+ viral particle clusters on the target cell surface (Fig. 5g), indicating that viral assemblies are rapidly transferred to target cells during cell contacts.
25 Finally, transmission electron micro scopy revealed Env+ mature viral particle clustered on the sides of cell contacts together with a mesh of electron-dense material, putatively, extracellular matrix (Supplementary Fig. 7a,c, arrows).
The inventors also observed Env viral particles in potential synaptic clefts (Supplementary Fig. 7b, arrowheads), although in a much lesser amount.

Therefore, during cell contacts, extracellular HTLV- 1 assemblies can be rapidly transferred outside the synaptic zone to the surface of other lymphocytes.

Relevance of extracellular viral assemblies in HTLV-1 infection To quantify the relevance of extracellular viral assemblies in HTLV-1 transmission, the inventors removed them by extensive pipetting or by competing the extracellular matrix with heparin. The inventors then measured virus transmission using reporter Jurkat T cells. (Fig. 6a). The treatments progressively reduced cell-associated Gag-p19, increasing Gag-p19 in cell supernatants, (Fig. 6b,c). Importantly, the capacity of heparin-washed cells to infect reporter cells was strongly diminished (= 80 %) and correlated with the amount of cell-associated Gag-p19 (Fig. 6d). Luciferase activity was due to virus infection, since it was inhibited by the reverse transcriptase inhibitor AZT (Fig. 6e). Cell supernatants from washed cells could infect reporter target cells, although much less efficiently, provided their previous concentration (Fig. 6f). Note that heparin-containing wash supernatants could compete for virus attachment on target cells. Mechanical and heparin treatments did not alter the capacity of cells to form cell contacts (Supplementary Fig. 8). Finally, similar results were obtained when HTLV- 1 transmission by T cells from HAM/TSP individuals was studied (Fig. 6g).

In conclusion, extracellular viral assemblies account for over 80 % of the infectious capacity of HTLV- 1 infected cells.

DISCUSSION
The inventors show that 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-cell-to-cell transmission. The present results are in line with previously reported data showing extracellular clusters of HTLV- 1 viral particles associated to electron dense material 37-39. Extracellular HTLV- 1 assemblies are strikingly reminiscent 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 spread upon fragmentation 13. Bacterial biofilms are rich in exopolysaccharides produced by bacteria 13 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 40-43 HTLV-1 hijacks similar host cell proteins enhancing their expression, modifying their carbohydrate composition, or their spatial organization to build extracellular assemblies. Interestingly, while collagen, fibronectin, galectin-1 and galectin-3 are overexpressed during HTLV-1 infection 23,24,32,33 the inventors found only collagen and galectin-3 accumulated in viral assemblies, indicating that preferential protein accumulation occurs to build these structures. Moreover, agrin, but not a-dystroglycan, accumulates in viral assemblies, although these two proteoglycans cluster at immunological synapses 26,28. Finally, HTLV-1 may utilize host cell adhesion molecules for attachment to the cell surface and for clustering at the uropod. It remains unknown, however, the site of virus budding into extracellular viral assemblies, and the contribution of cell-cell contacts to the formation or concentration of these structures. Therefore, extracellular matrix components and cellular lectins might together generate cocoon-type structures able to concentrate virions in a confined protective environment. Interestingly, hyperglycosylation of envelope glycoproteins may help viruses, including HIV1, to escape immune responses 20,44. Since HTLV-1 Env glycoprotein is poorly glycosylated, embedding viral particles in carbohydrate-rich supramolecular structures could help HTLV-1 to avoid immune recognition. Moreover, concentrating virions in these structures may locally increase "infectious titers" and help to convey the virus from cell to cell. Note worthy, extracellular composition and structure of extracellular matrix, including collagen and galectin-3, is modified by the 7-irradiation of cell cultures 45,46 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.

As reported 4,6,7, the inventors observed HTLV- 1 transmission via cell contacts. However, our data do not support a model of contact-induced virus budding and direct transfer through synaptic clefts, but rather, the existence of preformed transient extracellular structures that rapidly adhere to the surface of target cells during cell contacts. Nevertheless, the inventors also observed Env viral particles in the synaptic zone, but in a much lesser amount. The functional relevance of extracellular assemblies is definitely supported by the fact that removing these structures strongly inhibits HTLV-1 cell-to-cell transmission. Thus, although heparin washes did not completely remove extracellular viral assemblies, as assessed by fluorescence microscopy, the infectious capacity of cells was reduced by 80 %. Cell supernatants obtained after cell washes were infectious, although much less efficient, indicating that the integrity of extracellular viral assemblies and their transfer at cell contacts are key for HTLV- 1 transmission.

Extracellular viral assemblies were resistant to strong shear flow during extensive pipetting, suggesting that they can defy physiological fluid dynamics in vivo. Interestingly, irregular surfaces help the formation and stability of bacterial biofilms. Moreover, some bacteria induce host cell surface reorganization to resist shear flow 47. Similarly, 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. In vivo, viral assemblies might be rapidly transferred to other T lymphocytes or dendritic cells during cell contacts in secondary lymphoid organs. Dendritic cells, in turn, may get infected and/or transfer again HTLV-to T lymphocytes 2. In addition, dendritic cells could also process viral assemblies and present viral antigens to T lymphocytes, triggering and maintaining the anti-HTLV-1 immune response.

The evolutionary advantage of this transmission mechanism is at present unknown, but it might condition both virus spread and immune responses of HTLV-1 -infected subjects. It is likely that other viruses also developed transmission strategies based on similar biofilm-like viral assemblies. As extracellular structures of particular composition, biofilm-like viral assemblies might be potential targets for future anti-viral therapy.

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Claims (13)

1. A method of screening for a compound that affects formation of viral biofilm-like structure, comprising the steps of:

- contacting a cell with a virus naturally involved in viral biofilm-like structure and at least one candidate compound; and - identifying the compound that affects the formation of said viral biofilm-like structure or the cohesion of said biofilm.

2. A method of screening for a compound that affects virus transmission from cell to cell, comprising the steps of:

- infecting a cell with a virus naturally involved in viral biofilm-like structure for a time sufficient to form a viral biofilm-like structure;

- contacting said virus infected cell with a non-infected cell and at least one candidate compound; and - identifying the compound that affects transmission of said virus from the infected cell to the non-infected cell.

3. The method of claim 1 or 2, wherein the viral biofilm-like structure consists in a structure comprising carbohydrate rich matrix such as slex, adhesion molecules such as collagen and agrin and cellular linker proteins such as tetherin, galectine-3 and CD62.

4. The method of any one of claims 1 to 3, wherein the virus is a lymphotropic virus.

5. The method of claim 4, wherein the lymphotropic virus consists of HTLV, HIV.

6. A compound that affects formation and/or cohesion of virus biofilm-like structure obtained by the method of claim 1.

7. A compound that affects virus transmission from cell to cell obtained by the method of claim 2.

8. A composition comprising a compound as defined in claim 6 or 7, and a pharmaceutically acceptable carrier.

9. 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 a composition as defined in claim 8.

10. Use of a therapeutically effective amount of a composition as defined in claim 8 for preventing and/or treating a subject against a biofilm-like structure forming associated disease.

11. Use according to claim 10, wherein the subject is a human.

12. Use according to claim 11, wherein the subject is immunocompromised.

13. Use according to claim 10, wherein the biofilm-like structure forming associated disease is caused by HTLV-1 or HIV.