The remaining 4 mice were monitored for 14 days to record changes in bodyweight (b,e) and survival (c,f). oral immunizations with the influenza virus elicited significantly higher levels of virus-specific IgG and IgA antibody responses, as well as HAI titers in the sera. Upon challenge infection, the SL immunization elicited higher levels of pulmonary IgG antibody and CD8+ T cell responses than the oral immunization. Enhanced splenic germinal center B (GC B) and B cell proliferation were also detected from the SL immunization, both of which were significantly greater than those of the oral immunization. Importantly, compared to oral immunization, significantly lessened lung viral loads and bodyweight reductions were observed from the SL immunization and these parameters contributed to prolonging the survival of the immunized mice. These results indicate that both SL and oral administration could be effective routes in inducing protective immunity against influenza virus CDC25C infection, with SL immunization being the better of the two delivery routes. values of less than 0.05 were considered statistically significant (* 0.05, ** 0.01, and *** 0.001). 3. Results 3.1. Lethal Dose of Influenza Virus Administration by SL or Oral Route Showed No Virus Replication and No Bodyweight Loss The mice were inoculated with either low (100 PFU) or high (500 PFU) doses of live influenza viruses through SL or oral routes in order to confirm whether viral infection through these modalities is possible. At four dpi, lung tissues were collected and viral titer, as well as bodyweight changes, were compared to the IN positive control mice. Neither the SL nor the oral administration of the low dose of live influenza virus resulted in viral replication in the lungs, whereas lung virus titers were detected in the IN positive control group (Figure 1a). Consistent with this finding, the bodyweight changes were negligible in the SL and oral groups, whereas a gradual bodyweight reduction was observed in the IN positive control group over the first few days (Figure 1b). However, the IN mice eventually regained their bodyweight and none of the mice in all three of the groups perished (Figure 1c). Similar to the low dose of infection, the high dose of infection through the SL or oral routes did not elicit lung viremia. The viral Azathramycin titer elicited by a high dose of influenza virus inoculation through the IN route was comparable to that of the low dose of infection (Figure 1d). However, intranasally administering a high dose of influenza virus resulted in the deaths of mice by eight dpi, while all of the mice belonging to the SL and oral groups survived (Figure 1e,f). These results have demonstrated that oral or SL virus exposure does not incur viral replication in the lungs, regardless of infection dose, thereby confirming that these administration routes are safe. Open in a separate window Figure 1 Oral or sublingual administration with a lethal dose of live influenza virus were safe. Mice (n = 8 per group) were immunized with live influenza virus (A/Hong Kong/1/1968 (H3N2) with either 100 PFU or 500 PFU by oral, SL, or IN routes. From each group, 4 mice were sacrificed 4 days after virus inoculation and lung virus titers were determined (a,d). The remaining 4 mice were monitored for 14 days to record changes in bodyweight (b,e) and survival (c,f). Data are expressed as mean SD from four independent Azathramycin experiments. 3.2. SL or Oral Administrations Elicited Virus-Specific Antibody Responses and HAI Titer At three weeks after live virus administration, sera were collected in order to determine virus-specific antibody responses and HAI titer. Virus-specific IgG antibody responses elicited upon SL or oral live virus immunization were significantly greater than that of the unimmunized control group (Figure 2a). SL and oral immunization also induced virus-specific IgA antibody responses, albeit being induced Azathramycin to a much lower extent than the IgG levels (Figure 2b). As expected, HAI titers were not detected from the na?ve control, however, the SL and oral immunization induced HAI titers ranging between 60 and 80 (Figure 2c). While both oral and SL immunization induced antibody responses and HAI titers, significant differences between the two groups were not detected. Compared to these two administration routes, IN.