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

Figures

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  Figure A1. Analysis of guinea pig anti-RV serum specificity. Western blot analysis of whole HepG2 cell lysates from sWa[VP2/6/7] or GFP amplicon vector transduced (MOI 2) or non-transduced mock cells. Proteins were analyzed using the guinea pig polyclonal anti-RV antibody (upper panel); anti-actin antibody (middle panel); or the anti-GFP antibody (lower panel). Expected migration lengths of the RV proteins VP2 and VP6, actin and GFP are indicated on the left with arrows and the positions of the molecular weight markers are shown on the right in kDa.

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  Figure 1. Rotavirus (RV) protein synthesis upon herpes simplex virus type-1 (HSV-1) amplicon vector transduction. (A) Schematic representation of the polycistronic gene expression cassettes packaged into herpesvirus amplicon particles. Herpes simplex virus type-1 amplicon particles deliver a DNA cassette comprising a single polycistronic messenger RNA containing the coding sequences of three RV capsid proteins VP2, VP6 and VP7 separated by internal ribosome entry sites (IRES) and controlled by the HSV-1 immediate early (IE) 4/5 promoter. The HSV-1 origin of DNA replication (oriS) and packaging/cleavage signal (pac) as well as the SV40 polyadenylation sequence (polyA) are indicated. The synthetic RV cassettes derived from the human Wa strain were adjusted to human codon usage (synthetic strain Wa, sWa). The RRV[VP7/6/2] was derived from the rhesus strain RRV and encodes, in addition to the RV proteins, for the enhanced green fluorescent protein (EGFP) as reporter gene. The sWa[VP2/6/7_V5] amplicon vector harbors a C-terminal V5 tag at VP7. The GFP amplicon vector encoding EGFP was used as control vector; (B) Rotavirus gene expression from codon optimized transgene cassettes. HepG2 cells…

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  Figure 2. Ultrastructural analysis of transduced cells using electron microscopy. (A,B) Transmission electron micrographs of RVLP formation in the cytoplasm of vector-transduced cells. HepG2 cells were transduced (multiplicity of infection, MOI 5) with the HSV-1 amplicon vector sWa[VP2/6/7]. The cells were harvested 24 hpt from monolayers, processed as described in the material section and analyzed in a transmission electron microscope equipped with a charge-coupled device (CCD) camera; (B) Enlargement of the region marked in (A) showed clusters of regular circular structures found in the cytoplasm of the transduced cell. Scale bar: (A) 2 µm; (B) 0.5 µm. (C,D) Correlative light and electron microscopy. HepG2 cells were transduced (MOI 5) with the HSV-1 amplicon vector sWa[VP2/6/7_V5]. The cells were harvested 24 hpt from monolayers by pelleting and fixation with 4% formaldehyde and embedded in LR White. Ultrathin sections were directly collected on grids, prepared for immune fluorescence and images were taken using a fluorescence microscope. Thereafter, the same grids were further processed for electron microscopy. The fluorescent image was opened with the software Maps on the scanning…

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  Figure 3. Immune response in mice upon immunization with the vaccine vector sWa[VP2/6/7]. (A) Schematically represented in vivo studies as timeline. Before the immunization of female BALB/c mice a blood sample was collected from each animal. Five days after intra muscular (i.m.) injection of the prime dose, a male was added for mating. Before booster immunization, blood samples were taken. For the first set of experiments, mice received a booster immunization at 19 days after prime dose injection, and, for the second set of experiments, at 20 days after prime dose injection. Blood and milk samples were taken on a regular basis starting on the day of parturition. (B–D) Rotavirus specific immunoglobulin G (IgG) serum (B,D) or milk (C) levels of immunized mouse dams determined by enzyme-linked immunosorbent assay (ELISA). Rotavirus-specific IgG in sera (1:5000 dilution) or milk (1:500 dilution) of either sWa[VP2/6/7] or GFP amplicon vector immunized animals was measured and plotted relative to the positive control applied on each ELISA plate that was taken as 100%. As positive control always the same sera of a RV hyperimmunized mouse from a previous study [17] (diluted 1:5000) was used.…

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  Figure 4. Screening of mice sera using immune fluorescence show VP2- and VP6-specific antibodies in the sera of vaccinated dams. Vero2-2 cells transduced (MOI 1) with the corresponding monocistronic amplicon vectors encoding individual RV proteins VP2, VP6 or VP7 were stained 24 hpt by immune fluorescence. The sera of vaccinated dams served as primary antibodies (diluted 1:100) and were detected by the secondary antibody anti-mouse conjugated with Alexa-Fluor 594 (red). GFP fluorescence (green) expressed as reporter gene from each amplicon vector served to identify transduced cells, DAPI was used to stain the nuclei (blue). Polyclonal anti-RV serum served as positive control.

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  Figure 5. Analysis of rotavirus-like particles (RVLPs) produced in HSV-1 amplicon transduced cells. (A,B) electron micrographs of HSV-1 amplicon vector encoded RVLPs. HepG2 cells were infected with the polycistronic HSV-1 amplicon vector sWa[VP2/6/7] and two days after transduction, RVLPs were purified over a sucrose cushion and the concentrated particles were analyzed by transmission electron microscopy. (A) Negative staining of RVLPs from HepG2 cells transduced with sWa[VP2/6/7]. (B) Immunogold staining of the same sample of RVLPs as in (A) using a polyclonal anti-RV serum as primary antibody and a secondary antibody coupled to 12 nm colloidal gold particles. Scale bar: 100 nm (A,B). (C) Double-layered purified RVLPs and remaining RV proteins in the cell pellet. Vero2-2 cells were transduced with the indicated HSV-1 amplicon vectors (MOI 2) or non-transduced (mock). Cells were harvested 48 hpt and RVLPs were purified and concentrated from the supernatant over a sucrose cushion. The concentrated RVLPs (purified RVLPs, left panel) and the RV proteins remaining in the cell pellet obtained during RVLP harvesting (remaining pellet, right panel) was analyzed by Western blotting using a…