Figure 1.
Electron microscopy images of the single antigens and the trivalent combination used to immunize BALB/c mice.
Morphological assemblies of NoV GII-4 capsid (A), GI-3 capsid (B) and RV rVP6 (C) proteins, and the trivalent combination (1:1:1 of each antigen) of the structures depicted in panels A-C (D) were examined by transmission electron microscopy (TEM) using a FEI Tecnai F12 electron microscope (Philips Electron Optics, Holland) with 18,500 × magnification following negative staining with 3% uranyl acetate (UA), pH 4.6. The arrows represent each structure (A–C) in the trivalent assembly (D). Bar 100 nm.
Figure 2.
Antigen-specific serum IgG dose response.
Mice were immunized twice at study weeks 0 and 3 with 3, 10 and 30 µg of single antigens and the geometric mean titers (GMTs) induced by GII-4 VLPs (A), GI-3 VLPs (B) and RV rVP6 (C) were measured in an ELISA. The error bars represent the standard error of the means. Statistical differences between any two experimental groups were determined by a Mann–Whitney U-test and the p-value ≤ 0.05 was considered a statistically significant difference.
Figure 3.
Serum IgG responses induced by the single antigens versus trivalent combination.
Mice were immunized two times with 10 µg of the single antigen (GII-4 VLPs, GI-3 VLPs or rVP6) or the trivalent combination (each antigen at a 10 µg dose), and the sera at study week 5 and 24 were tested against GII-4 VLPs (A), GI-3 VLPs (B) and rVP6 (C) in ELISA. Shown are the geometric mean titers (GMTs) of the sera with standard errors of the means. The horizontal lines on panels A-C indicate the limit of detection for the assay. Kinetics of GII-4, GI-3 and VP6-specific IgG responses induced by the trivalent vaccine were measured from tail blood samples of immunized and control mice, and the OD values representing the quantity of antigen-specific IgG at any given time point are shown (D). The avidity of GII-4 (E), GI-3 (F) and rVP6-specific (G) serum IgG antibodies was tested from individual mouse termination sera (at 1:200 dilution) in a modified ELISA in which urea was used to strip off the low avidity antibodies. Shown are the individual mice antigen-specific avidity indexes (%) and the boxed values at the bottom of each figure indicate the group mean avidity indexes. The avidity index was calculated as (OD with urea/OD without urea) × 100%. Avidity index ≥ 50% was considered high avidity. Statistical differences between any two experimental groups were determined by a Mann–Whitney U-test and the p-value ≤ 0.05 was considered a statistically significant difference.
Figure 4.
Cross-reactive serum IgG antibodies.
Mice were immunized two times with 10 µg of the single antigen (GII-4 VLPs, GI-3 VLPs or rVP6) or the trivalent combination (each antigen at a 10 µg dose) and the sera were tested against heterologous NoV VLPs (A) and RV cell culture antigens (B) in ELISA. Shown are experimental and control groups’ mean OD values representing the quantity of antigen-specific IgG. The error bars represent standard errors of the mean. A Mann–Whitney U-test was used to determine statistical differences between single antigen-induced IgG quantities compared with trivalent vaccine induced IgG quantities at study week 5 and IgG quantities induced by the trivalent vaccine at study weeks 5 and 24. The p-value ≤ 0.05 is considered a statistically significant difference.
Figure 5.
Group-wise pooled stool samples (10% suspension) of mice immunized with the single NoV GII-4 VLPs, GI-3 VLPs or RV rVP6 antigens or the trivalent combination vaccine were titrated two-fold and anti-GII-4 (A), anti-GI-3 (B) and anti-VP6 (C) IgG content was measured in ELISA. Anti-VP6 IgG and IgA antibodies were measured from the trivalent combination vaccine immunized and control mice vaginal wash samples diluted 1:5 for IgG detection and 1:2 for IgA detection (D). Shown are experimental and control groups’ mean OD values representing the quantity of antigen-specific antibody.
Figure 6.
Functionality of NoV and RV-specific antibodies.
Termination sera of mice immunized with the single NoV GII-4 or GI-3 VLPs antigens or the trivalent combination vaccine were pooled group-wise, titrated two-fold and used for blocking the binding of homologous GII-4 and GI-3 VLPs (A, B) or heterologous GII-4 NO and GI-1 VLPs (C, D) to human secretor positive saliva (type A for GII-4, GII-4 NO and GI-3 binding and type O for GI-1 binding). Serum from mice receiving the carrier only (PBS) was used as a negative control. The blocking index (%) was calculated as 100% – (OD wells with serum/OD wells without serum, maximum binding) × 100%. Vaginal washes of mice immunized with the trivalent combination vaccine were tested for inhibition of human RV Wa (G1P1A [8]) strain homologous to the immunizing rVP6 protein, or rhesus RV (G3P5B [3]) infectivity by neutralizing ELISA (NELISA). Vaginal washes of mice receiving the carrier only (PBS) and serum from a RV seropositive human donor were used as negative and positive controls. Results are shown as the mean percentage (%) inhibition of rotavirus infectivity of duplicate wells with standard errors. A dashed horizontal line indicates 60% reduction in virus infectivity.
Figure 7.
NoV and RV-specific IFN-γ responses.
Splenocytes of mice immunized with the single NoV GII-4 or GI-3 VLPs or the trivalent combination vaccine were stimulated with synthetic NoV capsid-derived 15-mer peptides from different NoV genotypes and analyzed for IFN-γ production by an ELISPOT assay (A). The mean spot forming cells (SFC)/106 cells are shown. The error bars represent the standard errors of the mean. The statistical differences between any two experimental groups’ response to a given peptide were determined by a Mann–Whitney U-test and the p-value ≤ 0.05 was considered a statistically significant difference. Splenocytes of rVP6 or the trivalent vaccine immunized mice were pooled group-wise and stimulated with synthetic VP6-derived 18-mer peptide or RV cell culture antigens and analyzed for IFN-γ production by the ELISPOT (B). Splenocytes from mice receiving the carrier only (PBS) were used as negative control cells. The mean spot forming cells (SFC)/106 cells of the replicate wells are shown. The dashed line in each figure indicates the maximum background level (cut-off limit) obtained from cells incubated in a culture media (CM) only (mean SFC/106 + 3× SD).