Ab272504

Conventional immunohistochemistry (IHC) was performed on 3 µm thick, consecutive sections of FFPE samples with the Bond Polymer refine detection Kit (Leica, DS9800) according to the manufacturer’s instructions on a fully automated IHC stainer (Leica Bond). For IHC staining, the anti-HLA-G monoclonal antibody (mAb) (Abcam, UK, clone 4H84) was used as recently described [22 (link)]. In addition, mAbs directed against CD3 (Labvision, Germany, clone SP7), CD20 (DAKO, California, USA, clone L26), CD56 (Cell marque, Massachusetts, USA, clone MRQ-42) and the SARS-CoV nucleocapsid protein (Rockland Inc., Pennsylvania, USA, clone 200-401-A50) and the spike protein (Abcam, UK, clone ab272504) were employed according to the supplier’s instructions. Sections were examined and imaged with a Zeiss Axiophot microscope (Zeiss, Jena, Germany). Two pathologists (MB and CW), independently and blinded to the clinical data, scored all samples. HLA-G expression was analyzed using a Histoscore as previously described [65 (link)]. The relative amount of positively stained cells (%) was multiplied by their intensity from 0 (negative), 1 (weak), 2 (moderate) to 3 (intense) leading to the expression intensity (or H-score) that was further classified as absent (0), low (1–100), intermediate (101–200), or strong (201–300) overall expression.

Cell lysates were harvested in Laemmli sodium dodecyl sulfate–polyacrylamide (SDS) gel electrophoresis sample buffer containing 2% SDS and 5% β-mercaptoethanol. The lysates were boiled and loaded onto a polyacrylamide gel. The samples were separated on 12% resolving and 5% stacking SDS-PAGE gels in a mini electrophoresis unit (Bio-Rad, USA) at 100 V for 1 h. The proteins were transferred onto a nitrocellulose membrane (Millipore, USA) under wet conditions using a trans-blot apparatus (Bio-Rad, USA). After blocking with 5% skimmed milk, the membrane was incubated either with a rabbit polyclonal to SARS-CoV-2 spike glycoprotein (1/1000) (Abcam; ab272504) or a human antibody to the SARS-CoV-2 nucleocapsid protein (1:2500) (GenScript; HC2003) followed by a goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (1:2000 dilution, Invitrogen; USA) and a goat anti-human horseradish peroxidase (HRP)-conjugated antibody (1:2000 dilution, Invitrogen; USA), respectively. B-actin was used as a loading control in Western blot. The membrane was reacted with the ECL substrate solution (Pierce ECL, USA). The membrane was exposed to an autoradiograph film (KODAK X-OMAT, Sigma Germany), and was developed using a Kodak developer (X-OMAT 1000A, Sigma Germany).

The following primary antibodies were used: mouse monoclonal antibodies against MHV N and S proteins (kind gifts of Dr Helmut Wege, University of Würzburg), rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (ab272504, Abcam) mouse anti-GAPDH (IgM specific, G8795, Sigma-Aldrich), mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-HA (3724, Cell Signaling Technology), rabbit anti-PERK (ab229912, Abcam), rabbit anti-HERPUD1 (ab150424, Abcam), rabbit anti-GRP78 (BIP, ab108613, Abcam), rabbit anti-eIF2α (9722, Cell Signaling Technology), rabbit anti-phospho-eIF2α (Ser51, 9721, Cell Signaling Technology), rabbit anti-ATF4 (10835-1-AP, Proteintech), rabbit anti-ATF6 (ab203119 and ab37149, Abcam), mouse anti-S6 (2317, Cell Signaling Technology) and rabbit RPL10a (ab174318, Abcam). Secondary antibodies used for western blotting were purchased from Licor: IRDye 800CW Donkey Anti-Mouse IgG (H+L), IRDye 800CW Donkey Anti-Rabbit IgG (H+L), IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 680RD Goat Anti-Mouse IgM (μ chain specific).

Samples were resolved on Bolt^TM 4–12% Bis-Tris Plus gradient gels (Invitrogen, Waltham, MA, USA). Gels were either stained with Coomassie brilliant blue or subjected to Western blot using appropriate antibodies: rabbit polyclonal anti-SARS-CoV-2 S antibody (Abcam ab272504, Cambridge, UK) or rabbit polyclonal anti-SARS-CoV/CoV2 M antibody (Novus Biologicals NB100-56569, Littleton, CO, USA).

LION/repRNA potency was assayed in vitro. Briefly, serial dilutions of LION/repRNA were incubated on a monolayer of BHK cells in a 96-well plate. Twenty-four hours later, cell lysates were added to an ELISA plate coated with anti-SARS-CoV2 Spike (S1 domain) monoclonal antibody (Genetex GTX632604). Following a primary incubation and washes, a polyclonal anti-SARS-CoV2 Spike (full-length S) primary antibody was added (Abcam Ab272504). Following a secondary incubation and washes, a secondary horse radish peroxidase (HRP)-conjugated antibody was used to detect S-specific binding (Genetex GTX213110-01). Following a final incubation, HRP activity was assayed by TMB/HCL detection and absorbance measured by plate reader (EL_X808, Bio-Tek Instruments Inc) at 450nm.