3). Previous data suggested that the mucosal immunity induced by parenteral delivery of VRP may be due

to the induction of a mucosal-like environment in the draining lymph node [29]. It is therefore possible that there are long-term effects in the lymph node after prime with VRP which affect the immune response during boost. In addition, it was unknown whether VRP play a more important role during prime or boost, or if both are equally important. We addressed these questions in mice which received a prime and boost with OVA in the footpad. These mice were divided into groups which received no VRP, VRP in prime only, VRP Alectinib research buy in boost only, VRP in both prime and boost, or VRP and OVA in the contralateral footpad during boost. We performed these studies in the footpad instead of i.m., so that the injection would drain to a single lymph node, focusing any effects. A low VRP dose (103 IU) was used to minimize possible effects of VRP in other lymph nodes. Evaluation of anti-OVA IgG in the serum and IgA in fecal extracts demonstrates

a comparable adjuvant effect in mice receiving the boost in the same or contralateral footpad as in prime (Fig. 4A and B). The OVA-specific IgG titer was similarly increased when VRP was included in prime only (Fig. 4A). However, when VRP was present only in the boost, the IgG titer was increased but to a significantly lower level. A more dramatic effect was observed for fecal anti-OVA IgA. Mice receiving VRP only during the boost had no detectable anti-OVA IgA, but addition of VRP to the prime only induced an www.selleckchem.com/btk.html IgA response comparable to that seen in mice immunized with VRP during both prime and boost (Fig. 4B). After prime with antigen and VRP, we do not observe any mucosal response without an antigen boost (data not shown), so it is apparent that boost with antigen is still required to generate an IgA response. Previous studies

of VRP activity have evaluated events in the draining lymph node after boost [29]. Since the events which occur during prime are clearly crucial for the VRP adjuvant activity (Fig. 4), we examined the cytokine environment in the draining lymph node 6 h after prime with VRP by multiplex very analysis. We again used footpad rather than intramuscular injection because this route allows us to focus our analysis on a single lymph node. We first measured levels of 30 cytokines in draining popliteal lymph nodes harvested 6 h after footpad injection with OVA (10 μg) or with OVA combined with 104 IU VRP. In VRP-injected mice we observed a significant increase in 18 of the 30 measured cytokines (Table 1). Based on the collected data, we selected a panel of 10 cytokines, 9 of which had a large fold increase in response to VRP, and one negative control (IL-12p40). We assessed those cytokine levels after injection of OVA alone or OVA with a range of VRP doses between 101 and 105 IU.