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.

Previous work showed that VP7 might play a key role in passive protection of pups from RV-induced diarrhea [27] . Based on the results from the immune fluorescence analysis of the mice sera, we were able to raise VP2-and VP6-specific antibodies in the blood of sWa[VP2/6/7] vaccinated dams, but no VP7-specific antibodies. The use of HSV-1 amplicon vectors to launch the in situ production of RVLPs provides the advantage that as opposed to injection of purified VLPs, the delivery of VLP encoding genes into host cells results in the intracellular production of antigens. In our case, we expected the formation of triple-layered RVLPs. Therefore, we further characterized the nature of our antigen used for vaccination. In a first step, RVLPs formed upon transduction of sWa[VP2/6/7] gene expression cassettes was analyzed using electron microscopy ( Figure 5A,B) and in a second step, the composition of purified particles was examined using Western blot analysis ( Figure 5C ). Negative staining transmission electron microscopy of the purified cell lysates from sWa[VP2/6/7] transduced cells showed that the synthesized RV structural proteins assembled into RVLPs similar in size and structure to wild-type RV particles [28] . Further immunogold labeling of these RVLPs using the rabbit polyclonal anti-RV serum and a secondary antibody conjugated to gold particles confirmed the VLP's identity as RV-like. sWa[VP2/6/7] transduced cells showed that the synthesized RV structural proteins assembled into RVLPs similar in size and structure to wild-type RV particles [28] . Further immunogold labeling of these RVLPs using the rabbit polyclonal anti-RV serum and a secondary antibody conjugated to gold particles confirmed the VLP's identity as RV-like. To analyze the composition of the isolated RVLPs, Western blot analysis of the purified RVLPs was performed ( Figure 5C ). This indicated that the observed RVLPs consisted predominantly of the two inner layers VP2 and VP6 ( Figure 5C, left panel) , whereas the outer layer protein VP7 remained in the cell debris pellet obtained during the purification of the RVLPs ( Figure 5C, right panel) . Moreover, VP7 is a transmembrane glycoprotein localized at the ER [29] and seemed to localize with the ER membranes ( Figure 1D ) even though VP2 and VP6 were co-expressed. In summary, even though VP7 was abundantly present in transduced cells ( Figure 1B,C) , it was hardly incorporated into the isolated RVLPs leading to the conclusion that in our system double-layered RVLPs were preferentially formed.