After purification, the absence of detectable replication-competent virus was confirmed by cytopathic effect assay, and VRP were titered by infection of BHK-21 cells as measured by immunofluorescent staining of VEE non-structural proteins. VRP genome equivalents (GE) were determined by RNA extraction with an Ambion MagMAX Viral RNA Isolation Kit followed by real time PCR using nsP1-specific primers and probe as previously described [27]. The ratio of VRP GE to BHK infectious unit (IU) was approximately 103. Six- to eight-week-old female Balb/c or C57Bl/6 mice

were purchased from Charles River and were housed at the University of North Carolina Division of Laboratory Animal Medicine animal facility according to protocols approved by the Institutional PI3K cancer Angiogenesis inhibitor Animal Care and Use Committee. Balb/c mice were used for all experiments except for assay of T cell responses to OVA, for which C57Bl/6 mice were used. Mice were injected in the rear footpad or by intramuscular delivery on weeks 0 and 4 with chicken egg albumin (OVA) (Sigma) (10 or 100 μg) alone or OVA mixed with the stated infectious units (IU) of VRP, as described in the text. Endotoxin in the OVA preparation was reduced below

the level of detection by phase separation using Triton X-114 [28]. For some experiments, OVA was conjugated to Alexa Fluor 488 using the Alexa Fluor 488 Protein Labeling kit (Invitrogen). Serum was collected from mice 3 weeks after boost. For isolation of fecal extracts, fecal pellets were collected 10 days after boost and vortexed at 4 °C at 0.2 g/ml in PBS containing 10% goat serum and 1× protease inhibitors (Roche) until pellets were disrupted. Samples were centrifuged, and supernatants were filtered through 0.22 μm filters OVA-specific IgG and IgA antibodies Amisulpride were detected by ELISA on 96-well high binding plates (Thermo Scientific) coated

with 10 μg/ml OVA in PBS. Sera and fecal extracts were added to plates in serial dilutions. OVA-specific antibodies were detected with horseradish peroxidase conjugated antibodies specific for mouse IgG (Sigma) or mouse-IgA (Southern Biotechnology) followed by addition of o-phenylenediamine dihydrochloride substrate (Sigma) for 30 min. Endpoint titers were determined as the last sample dilution that generated an OD450 reading of greater than 0.2. For determination of total IgA levels in fecal extracts, 96-well plates were coated with 5 μg/ml rabbit anti-mouse-IgA (Invitrogen), ELISAs performed as above, and a standard curve generated from dilutions of purified murine IgA (Sigma). This standard curve was used to determine the concentration of both OVA-specific and total IgA in fecal extracts. Mice were immunized in the footpad with either 10 μg OVA, or OVA + VRP.