Transfer of Anti-Rotavirus Antibodies during Pregnancy and in Milk Following Maternal Vaccination with a Herpes Simplex Virus Type-1 Amplicon Vector

Meier, Anita F.; Suter, Mark; Schraner, Elisabeth M.; Humbel, Bruno M.; Tobler, Kurt; Ackermann, Mathias; Laimbacher, Andrea S.

Cells were transduced with the indicated amplicon vectors (MOI 2) and total cell lysates were harvested 24 hpt or 48 hpt if RVLPs were purified. Either whole cell lysates or sucrose purified RVLPs (see section "RVLP purification") were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose membranes, probed with primary antibodies, and stained using anti-mouse (Sigma-Aldrich, Buchs, Switzerland) or anti-rabbit (Southern Biotech, Birmingham, AL, USA) IgG antibodies conjugated with horseradish peroxidase (HRP), followed by detection with WesternBright ECL spray ( directly labeled with Zenon Alexa-Fluor 594 according to the manufacturer's instructions (Molecular Probes, Thermo Fisher Scientific), mouse monoclonal anti-VP6 (1:500; Novus Biologicals, Cambridge, UK) and the anti-mouse Alexa-Fluor 633 (1:500; Molecular Probes, Thermo Fisher Scientific), mouse monoclonal anti-V5 antibody directly labeled with fluorescein isothiocyanate (FITC) (1:500; Molecular Probes) for the V5-tagged VP7. The ER was stained using the Alexa-Fluor 405 conjugated lectin ConA conjugated with Alexa Fluor 594 (20 µg/µL in PBS; Molecular Probes). Cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL in PBS, Roche, Basel, Switzerland) to visualize nuclei. After washing the cells with PBS and H 2 O, the coverslips were mounted in ProLong Gold (Molecular Probes). Samples were analyzed using a confocal laser-scanning microscope SP8 (Leica Microsystems, Wetzlar, Germany, 63× oil objective (numerical aperture (NA) = 1.40)). For analysis of RV protein-specific antibodies in the sera from immunized mice, sera was diluted 1:100 and detected by staining with a secondary anti-mouse antibody conjugated with Alexa-Fluor 594 (1:500; Molecular Probes). Pictures were taken using the fluorescence microscope (Axio Observer inverted microscope, Carl Zeiss AG, Oberkochen, Germany).