Delayed-type hypersensitivity (DTH) reaction to rotavirus EDIM and RRV
To analyze RRV- and EDIM-specific cell-mediated immunity of all study groups, a DTH response was measured by subcutaneous injection of rotavirus in the ear pinnea, RRV CCID50 of 2.5 × 107 in the right ear and EDIM 4 μg/ml in the left ear. As a control, the same rotaviruses were also administered to non-inoculated mice (n = 15) of the same age. There was little measurable swelling compared to the control with RRV (data not shown). It has been shown previously that a rotavirus-specific DTH response was not elicited when neonatal mice were infected at any time point after primary infection. EDIM induced a small but significant (p = 0.01) increase in ear swelling in the EDIM group (group B) compared to the control, indicating the induction of specific cellular immunity. The RRV (group A), RRV+EDIM (group C) and Gastrogard-R® (group D) groups were all comparable to the control. This might indicate that mice who received a primary infection at a young age show a DTH suppression when re-infected at an older age.
In vitro Concanavalin A and rotavirus-specific proliferation in spleen cells
To analyze T cell responses, spleen cells from all study groups were isolated and ex vivo restimulated with either Concanavalin A (Con A), RRV or EDIM. As a control, spleen cells of non-inoculated mice (n = 15) of the same age were used. Con A induced a significant stimulation of T cell proliferation with an average stimulation index (Con A stimulated cells/non-stimulated cells) of approximately 30. However, there were no differences seen between the groups (data not shown). The RRV+EDIM group (group C) as well as the Gastrogard-R® group (group D) showed a significant increase in T cell proliferation (p < 0.05 and p < 0.0001 respectively) compared to non-infected mice. These results suggest that multiple infections are needed to acquire a sufficient amount of rotavirus-specific memory T cells in the spleen to be able to re-stimulate these T cells in vitro. Rotavirus-specific serum IgM and IgG antibodies
Rotavirus-specific IgM and IgG were measured in the serum individually collected at day 16 and then pooled per group (pre-EDIM serum) and in the individual sera of the pups collected at day 28 (post-EDIM serum). RRV inoculation at day 7 resulted in rotavirus-specific IgM titers (Figure 6A) in the pre-serum of about 100 AU in the RRV and RRV+EDIM groups. There was no rotavirus-specific IgM detectable in the EDIM group (group B). Surprisingly, even though clinical symptoms during the RRV infection were inhibited by Gastrogard-R® (group D), a low rotavirus-specific IgM level (10 AU) was measured in this group. In the post EDIM inoculation sera, the rotavirus-specific IgM titers of the RRV and RRV+EDIM groups were not different from the pre-serum, but the rotavirus-specific IgM antibody titer was increased in the EDIM group as well as the Gastrogard-R® group to 90 AU.