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SUPPLEMENTARY MATERIAL

Table of Contents

Confirmatory study of anti-Env monoclonal antibodies specificity on human transfected cells with plasmid encoding HERV-W MSRV-Env protein

A. MATERIALS AND METHODS............................................................................................2

Human cell cultures.........................................................................................................................2

Plasmids...........................................................................................................................................2

Cellular transfections.......................................................................................................................2

Immunofluorescence and confocal microscopy...........................................................................3

Paraffin embedding of pelleted cells and Immunohistochemistry.............................................3

B. RESULTS..................................................................................................................................4

Immunocytochemistry (fluorescence detection)..........................................................................4

Immunohistochemistry on sections from paraffin-embedded cell pellets (DAB detection)....4

C. FIGURES..................................................................................................................................5

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Confirmatory study of anti-Env monoclonal antibodies specificity on human transfected cells with plasmid

encoding HERV-W MSRV-Env protein.

A. MATERIALS AND METHODS

Human cell cultures

HEK-293T, transformed human embryonic kidney cells, were cultured in DMEM (Dulbecco's

modified Eagle's medium high glucose, Invitrogen) with 10% Fetal Calf serum (CFS), 1%

penicillin/streptomycine, 1% no- essential amino-acids (NEAA) and 1% sodium pyruvate (Na-

py). THP-1, human monocyte cells derived from acute monocytic leukemia, were cultured in

RPMI (Roswell Park Memorial Institute, Invitrogen) 1640 with 10% Fetal Calf serum (CFS),

1% penicillin/streptomycine and 0,05mM β-mercaptoethanol.

Plasmids

pMax-GFP (Invitrogen), encoding GFP (green fluorescent protein) reporter gene controlled

by pCMV (Cytomegalovirus promoter). This plasmid was used to optimise conditions for

transfections in the cell lines. pCMV-env-ms (GeNeuro), HERV-W env gene sub-cloned from

pV14 plasmid (encoding MSRV-Env protein), itself cloned from cDNA of RNA extracted from

purified virus particles in supernantants of MS cell cultures (MSRV) controlled by pCMV [1,2].

Cellular transfections

Optimal transfection conditions (80% efficiency) for HEK-293T cells were obtained with

Lipofectamine 2000 (Invitrogen). After 24h culture of newly seeded cells, 80 à 90%

confluency was reached as appropriate for transfection. Plasmid is mixed with lipofectamine

in Opti-MEM Reduced Serum medium (Gibco, ref.51985-034), incubated at room

temperature for 20 min, then added onto the cell culture and further incubated at 37°C, with

5% de CO2 for 72h. Optimal transfection conditions (about 20% efficiency, compared to 0%

with lipofectamine) for THP-1 cells were obtained by electroporation with Amaxa nucleofector

(Lonza). 2 millions cells were pelleted by centrifugation and resuspended in 100µL of

Nucleofector and maintained 15 min at room temperature (RT) before 2µg of plasmid was

added. Electroporation was then performed with protocol V-001. After nucleofection, 500µL

of culture medium was added and cells were aliquoted in a 12-wells culture plaque and

adjusted to a final volume of 1mL medium per well. Cells were then incubated at 37°C with

5% CO2 for 24h.

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Immunofluorescence and confocal microscopy

Cells were washed 3 times in phosphate buffer saline (PBS) before 200μl of cell suspension

were deposited onto polylysine-coated slides (Roth) and incubated 40 min at room

temperature for cells to adhere. Slides were then washed twice with PBS and fixed for 10

min in 500μl paraformaldehyde (PFA) 3% solution at RT. Cells were washed 3 times in PBS

before incubation with antibodies or kept 1 to 3 days in PBS at 4°C in the dark. The following

antibodies were used, according to the experiments: GN-mAb_01 or GN-

mAb_03 (GeNeuro), anti-HERV-W/Env mouse IgG monoclonal antibody; anti-

IgG1 secondary antibody specific for murine IgG1 Fc chain, coupled to Alexa fluor 555

(Invitrogen A-21127).

For immunolabelling, cells were incubated with 500μL of permeabilization buffer (PBS

1X/Saponin 0.2%/BSA 2%) for 20 minutes at RT. Cells are then labelled in the

permeabilization buffer with GN-mAb_01/FITC (1μg/mL), and/or with GN-mAb_03 (1µg/mL)

and/or anti-mouse IgG1 Alexa 555 (3µg/mL) for 1 he at RT in the dark. Slides were washed

twice with permeabilization buffer and incubated for 5min in this buffer. Slides were then

dried and mounted with coverslip in mounting medium (Dako). They were taken for

microscopic examination and/or kept in the dark at 4°C for few days or at -20°C for longer

time. Confocal microscopy examination was performed with Biphoton microscope LSM 510

NLO META (Zeiss) or CONFOCOR2 (Zeiss) with 63x/1.4 lenses. Argon lasers (458nm,

477nm, 488nm, 514nm) and HeNe1 (543nm) were used.

Paraffin embedding of pelleted cells and Immunohistochemistry

Cells were pelleted by centrifugation, fixed with formalin and pellets further embedded in paraffin with the standard protocol.

Paraffin sections, approximately 4 µm thick, were prepared from paraffin embedded cell pellets on Superfrost Plus glass slide and let drying at least 1 hour in the oven at 45°C. The dried paraffin sections were stored at 4°C until used.

Antibodies used in the test:

Antibody Supplier Reference Batch Concentration

GN_mAb_Env03 Geneuro GN_mAb_Env03 SQ09AK513

5.469 mg/mL

Isotype mouse IgG2a

Abcam Ab18415 GR2289-11-3 0.5 mg/mL

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The GN_mAb_Env03 antibody, a mouse monoclonal antibody directed against the MSRV-Env protein (HERV-W family), was kept at -20°C. The mouse IgG2a isotype antibody was purchased and kept at 4°C in accordance with the product datasheet recommendations.

The detection kit UltraView DAB was used on the Benchmark automated platform for the chromogenic detection of the primary antibody (multimer based detection kit, containing the labelled anti-mouse IgG secondary antibody, for the detection of the specific primary antibody).

The validation was made with the positive cell pellet to test different pretreatments. Positive labeling was noted with 4 different pretreatments and without pretreatment. No nucleolar staining was seen in the cell pellets in all cases whereas one pretreatment showed faint non-specific membrane staining at highest concentrations of either specific primary antibody or its unrelated control isotype. Therefore, the IHC protocol without pretreatment, which did not yield any background staining in negative cells even at highest concentrations of the primary antibody (specific or Isotype control), was then applied to the positive and the negative pellets with a series of dilutions of the primary antibody (1, 2.5, 5 and 10 µg/mL).

The validation was finalized with the mouse IgG2a isotype antibody applied at a concentration of 10 µg/mL on both the positive and the negative cell pellets. The immunostained slides with the isotype antibodies showed no staining in the same selected conditions (no pretreatment).

B. RESULTS

Immunocytochemistry (fluorescence detection)

Two human cell lines, permissive for HERV-W envelope (MSRV-env) expression, were used

to study the specificity of MSRV-Env detection with mouse monoclonal antibodies. Similar

results were obtained with either GN-mAb_01 or GN-mAb_03, which confirmed the

specificity of the detection with two different antibodies. One of those (GN-mAb_03) had

already been validated as specific for MSRV-Env detection in MS brains in parallel with two

other monoclonal antibodies specific for distant MSRV-Env epitopes (GN-mAb-04 and _05)

[3].

In Figure S1, transfected HEK293-T cells show that the envelope protein is specifically

expressed and detected. It appears mostly localized at the membrane level (Fig.S1B). In

permeabilized THP-1 cells the envelope protein is also specifically and abundantly

expressed and mostly localized at the membrane level and in intracellular Golgi-like

structures (Figure S1C). No background staining was seen in mock transfected cells

(Fig.S1A).

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Immunohistochemistry on sections from paraffin-embedded cell pellets (DAB detection)Illustration of slides of the validation batch using optimal antibody concentration for the

present conditions (5µg/ml) are given in In Figures S2. The positive cell pellet with no

pretreatment and immunostained with GN-mAb_03 primary antibody is presented in the left

illustration; parallel results with the corresponding isotype control are represented in the right

illustration. Transfected HEK293-T cells showed that the envelope protein is specifically

expressed and detected in suitable conditions for immunohistochemistry with formalin-fixed

and paraffin-embedded tissues. It appears mostly localized at the membrane level, but is

also detected in the cytoplasm of several effectively transfected and expressing cells (Fig.

S2, left picture). No background staining was seen in transfected cells from the same positive

pellet with the isotype control antibody (Fig. S2, right picture). No staining at all was seen

with either antibody in mock transfected cells.

Of note, S2 picture definition allows zooming on regions of interest.

C. Figures

Figure S1:

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Figure S2:

Slide 15.11.0096B.002/01.012-GN_mAb_Env03 Slide 15.11.0096B.002/01.013-Isotype

REFERENCES: 1. Perron H, Jouvin-Marche E, Michel M, Ounanian-Paraz A, Camelo S, et al. (2001) Multiple sclerosis

retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation. Virology 287: 321-332.

2. Komurian-Pradel F, Paranhos-Baccala G, Bedin F, Ounanian-Paraz A, Sodoyer M, et al. (1999) Molecular cloning and characterization of MSRV-related sequences associated with retrovirus-like particles. Virology 260: 1-9.

3. Perron H, Germi R, Bernard C, Garcia-Montojo M, Deluen C, et al. (2012) Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease. Mult Scler 18: 1721-1736.