Skimmed milk powder

To examine the conservation of RSV antigenicity, dot blot analysis and enzyme-linked immunosorbent assays (ELISAs) were performed. ELISAs were performed as described with the same antibodies as dot blots (19 (link)–21 (link)). For dot blot, 2 µL of each inactivated RSV sample, with respective untreated controls, were put on a nitrocellulose membrane (GE Healthcare Life Sciences, Germany). The membranes were blocked with 5% (w/v) skimmed milk powder (Carl Roth, Germany) in PBS-T (PBS with 0.05% Tween 20) (Bio&Sell, Germany; Carl Roth) for 1 h and incubated with a monoclonal antibody (mAb) recognizing RSV-F [18F12, 1:400 (26 (link))], a mAb recognizing the prefusion form of RSV-F (RSV-preF) (D25, 1:1,000; Cambridge Biologics, USA), or a mAb recognizing RSV-G (8C5, 1:500; Invitrogen, USA) in 2% (w/v) skimmed milk in PBS-T at 4°C overnight. After washing three times with PBS-T, the membranes were incubated with the respective secondary antibody: for 18F12 and 8C5, the peroxidase AffiniPure™ sheep anti-mouse IgG (H+L)-horseradish peroxidase (HRP) antibody (Dianova, Germany) diluted 1:500, and for D25, the goat anti-human IgG HRP-conjugated 1:20,000 (Dianova) for 1 h. The membranes were washed, developed with enhanced chemiluminescent substrate (Pierce, USA), and imaged on a CELVIN^® chemiluminescent imager (Biostep, Germany).

Baker’s yeast S. cerevisiae (Deutsche Hefewerke GmbH, Nürnberg, Germany) was used. A sufficient amount of the same batch for all experiments was stored at −20 °C until use. For all experiments, cells were suspended in purified water, 1/5 × PBS (isotonic phosphate-buffered saline solution, pH 7.4) (Sigma-Aldrich Chemie GmbH, München, Germany) or purified water containing cryoprotectants, respectively. Cryoprotectants dextran 20, glycerin and skimmed milk powder were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany), l-Glutamic acid and α-Lactose monohydrate from Sigma-Aldrich Chemie GmbH (München, Germany), d(+)-Maltose monohydrate from AppliChem GmbH (Darmstadt, Germany) and trehalose dihydrate from intelligent sugar GmbH (Otzberg, Germany).
For determining colony forming units, yeast extract peptone dextrose (YPD) agar plates with 20 ${\mathrm{g}\mathrm{L}}^{-1}$ of peptone ex casein, 10 ${\mathrm{g}\mathrm{L}}^{-1}$ of yeast extract, 22 ${\mathrm{g}\mathrm{L}}^{-1}$ of glucose monohydrate and 15 ${\mathrm{g}\mathrm{L}}^{-1}$ of Agar-Agar Kobe 1 were used, all chemicals supplied by Carl Roth GmbH + Co. KG (Karlsruhe, Germany).
Direct compression excipients used were microcrystalline cellulose (MCC) (Vivapur 102, JRS Pharma GmbH + Co. KG, Rosenberg, Germany), dicalcium phosphate (DCP) (Emcompress Anhydrous, JRS Pharma GmbH + Co. KG, Rosenberg, Germany) and lactose (Tablettose 70, Meggle-Pharma, Wasserburg am Inn, Germany).