Combined Live Oral Priming and Intramuscular Boosting Regimen with Rotarix® and a Nanoparticle-Based Trivalent Rotavirus Vaccine Evaluated in Gnotobiotic Pig Models of G4P[6] and G1P[8] Human Rotavirus Infection

Figure 1. Study design to assess the immune response and protective efficacy of the vaccine regimens in Gn pigs. Pigs were vaccinated with two doses of vaccines on PVD 0 and 14. Blood/serum samples were collected on PVD 0, 14, 28, and PCD 7. The immunized pigs were challenged with virulent Wa HRV or Arg HRV on PCD 0 and rectal swab samples were collected daily post-challenge for seven days for the assessment of diarrhea and virus shedding.

Figure 2. Daily mean diarrhea scores from PCD 0–7 in Wa (A) and Arg HRV-challenged (B) Gn pigs. At PVD 28, Gn pigs were orally challenged with 1 × 105 FFU of either Wa or Arg HRV and monitored for diarrhea severity and duration via daily rectal swabs for 7 days. Differing letters indicate significant difference (two-way ANOVA of repeated measures through time, followed by Tukey’s multiple comparisons test; p ≤ 0.05), and error bars indicate SEM.

Figure 3. Protection from virus shedding as measured by CCIF in Wa- and Arg HRV-challenged Gn pigs. At PVD 28, pigs were orally challenged with 1 × 105 FFU of either Wa or Arg HRV and monitored for virus shedding via daily rectal swabs for 7 days. (A,E) Mean onset day of virus shedding in Wa or Arg HRV-challenged pigs; (B,F) Mean days with virus shedding in Wa or Arg HRV-challenged pigs; (C,G) Mean peak virus shedding titers in Wa or Arg HRV-challenged pigs; (D,H) Mean area under the curve of virus shedding in Wa or Arg HRV-challenged pigs. Asterisks indicate degree of statistically significant difference (ordinary one-way ANOVA; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Error bars indicate SEM or (geometric SD for peak titers).

Figure 4. Daily fecal virus shedding as measured by CCIF in prime-boost, trivalent 2x and control-vaccinated pigs from PCDs 0-7. Daily rectal swabs were taken for evaluating fecal virus shedding after challenge with Wa HRV. Infectious virus particles were measured by CCIF and results are expressed as FFU/mL. Fecal samples from mock-infected pigs were used as negative controls. Different capital letters above data points indicate significant differences between groups at the same time point, while shared letters or no letters indicate no significant difference, according to two-way ANOVA of repeated measures through time, followed by Tukey’s multiple comparisons test (p ≤ 0.05). Bars indicate SEM. CCIF, cell culture immunofluorescence; FFU, focus forming units. (A) Daily Mean CCIF Titers (Wa G1P[8]), (B) Daily Mean CCIF Titers (Arg G4P[6]).

Figure 5. P[8], P[6], and P[4]-specific serum antibody responses in Wa HRV-challenged Gn pigs. Serum was collected at PVD 0, 14, 28 (PCD 0), and 35 (PCD 7) to evaluate systemic P-type-specific IgG (A) and IgA (B) responses using direct ELISA. Asterisks indicate degree of statistically significant difference (mixed-effects analysis with Dunnett’s multiple comparisons test; * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001). Lines and error bars indicate mean and SEM, respectively.

Figure 6. P[8], P[6], and P[4]-specific serum antibody responses in Arg HRV-challenged Gn pigs. Serum was collected at PVD 0, 14, 28 (PCD 0), and 35 (PCD 7) to evaluate systemic P-type-specific IgG (A) and IgA (B) responses using direct ELISA. Asterisks indicate degree of statistically significant difference (mixed-effects analysis with Dunnett’s multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). Lines and error bars indicate mean and SEM, respectively.

Figure 7. Intestinal IgA antibody titers in vaccinated and control Gn pigs challenged with either Wa (A) or Arg HRV (B). At PVD 35/PCD 7, Gn pigs were euthanized and small intestinal contents were collected and evaluated using direct ELISA to detect P-type specific IgA antibody responses. Asterisks indicate degree of statistically significant difference (mixed-effects analysis with Tukey’s multiple comparisons test; * p ≤ 0.05). Error bars indicate SEM.

Figure 8. Serum virus neutralization of P[8], P[6], and P[4] HRV in Wa HRV (A) or Arg HRV-challenged Gn pigs (B). Serum was collected at PVD 0, 14, 28 (PCD 0), and 35 (PCD 7) and evaluated for neutralizing antibody activity. Differing letters indicate significant difference (mixed-effects analysis using two-stage linear step-up procedure, p ≤ 0.05), and error bars indicate SEM.

Figure 9. Total numbers (A) and frequencies (B) of P[8], P[6] and P[4]-specific IFN-γ-producing CD3+CD4+ and CD3+CD8+ T cells in blood, ileum and spleen of Wa HRV-challenged Gn pigs at PCD 7 detected by flow cytometry. MNCs were stimulated in vitro for 17h with P[8], P[6] and P[4] S60-VP8* antigen, or mock stimulated. Asterisks indicate degree of statistically significant difference (multiple Mann-Whitney tests; * p ≤ 0.05, ** p ≤ 0.01). Error bars indicate SEM.

Figure 10. Total numbers (A) and frequencies (B) of P[8], P[6] and P[4] type-specific IFN-γ-producing CD3+CD4+ and CD3+CD8+ T cells in blood, ileum and spleen of Arg HRV challenged Gn pigs at PCD 7 detected by flow cytometry. MNCs were stimulated in vitro for 17 h with P[8], P[6] and P[4] S60-VP8* antigen, or mock stimulated. Asterisks indicate degree of statistically significant difference (multiple Mann-Whitney tests). Error bars indicate SEM.

Figure 11. P-type specific antibody-secreting cell (ASC) responses in the ileum and spleen of Wa (A) or Arg (B) HRV-challenged Gn pigs at PCD 7. Asterisks indicate degree of statistically significant difference (Kruskal-Wallis test with Dunn’s multiple comparisons test; * p ≤ 0.05). Error bars indicate SEM. Only ASC responses with significant differences among the groups are presented.