A remarkable difference was observed in the type-specific avidities elicited by GII.4 1999 and GII.4 2012 VLPs (Figure 1b). GII.4 2012 immune sera only had low blocking activity against GII.4 2006 VLPs. Amino acid substitution in the NERK motif (amino acids 310, 316, 484, and 493, respectively), altering the access to conserved blocking epitope F, moderately improved the cross-blocking responses against mutated GII.4 2012 VLPs (D310N). NoV GII.4 1999 VLPs, uptaken and processed by antigen-presenting cells, induced AR7 stronger interferon gamma (IFN-) production from mice splenocytes than GII.4 2012 VLPs. These results support the use of GII.4 1999 VLPs as a major component of a NoV vaccine. for 10 min and suspended in CM containing 20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, Abcam, Cambridge, UK). BM-cells were seeded at 1106 cells/mL (10 mL per plate) in non-treated 90 14.2-mm sterile petri dishes (VWR, Radnor, PA, US) and cultured at 37 C, 5% CO2 for 8 days. Fresh CM with GM-CSF (5 mL/plate) was added on the dishes on days 4 and 7 and the cells were FZD6 harvested on day 8. The generated cells were surface stained with phycoerythrin (PE)-conjugated anti-mouse CD11c and Horizon Viability Stain 780 (both from BD) and acquired using BD FACSCanto II flow cytometer as described earlier  which confirmed the cells to be 90% CD11c+ cells. The BMDCs were frozen according to published procedure  in ice-cold CM containing 10% DMSO (Sigma-Aldrich). The BMDCs were thawed, washed twice (300 = 0.264) of type-specific IgG response, with GMTs of 102,400 and 86,100 (95% CI = 53,200C139,300), respectively. IgG responses against homologous VLPs were significantly higher ( 0.05) than cross-reactive responses induced by heterologous antigen. GII.4 1999 VLP immunization induced significantly higher (= 0.018) cross-reactive IgG response against GII.4 2006 VLPs (GMT 25,600, 95% CI = 9600C69,900) than GII.4 2012 VLP immunization (GMT 3200, 95% AR7 CI = 1500C7000). When GII.4 1999 and GII.4 2012 cross-reactive responses were compared against each other, GII.4 1999 immunization resulted in 2-fold higher GII.4 2012-specific titer (GMT 16,900, 95% CI = 5800C49,200) than GII.4 2012 immunization against GII.4 99 (GMT 8060, 95% CI = 3900C16,900) but the difference was not statistically significant (= 0.15). Control mice did not develop specific IgG response to any of the VLPs tested. Open in a separate window Figure 1 Titers and avidity of norovirus (NoV) type-specific and cross-reactive serum immunoglobulin G (IgG) antibodies. Mice were immunized with GII.4 1999 (5 mice) and GII.4 2012 (4 mice) virus-like AR7 particles (VLPs) and the immune sera was used in enzyme-linked immunosorbent assay (ELISA) to determine the magnitude of IgG antibodies against homologous and heterologous NoV VLPs (a). Serum of mice receiving phosphate buffered saline (PBS) (5 mice) was used as a negative control (Ctrl). Shown are the geometric mean titers (GMTs) with 95% confidence intervals (error bars) counted from individual mice end-point titers in each immunization group. The dashed line illustrates the cut-off titer for samples considered positive. The avidity of IgG antibodies was measured from individual mice sera against homologous and heterologous NoV VLPs (b) as described in the Material and Methods. Horizontal lines in the box plots represent the medians, cross-symbols () represent the means, and the boxes illustrate the interquartile range that contains 50% of values with whiskers extending to the highest and lowest values. The antigen-specific antibody titers and the avidity indexes between immunization groups were compared by the KruskalCWallis test and significant differences (value 0.05) are identified with an asterisk (*). Comparison of the avidity of type-specific and cross-reactive antibodies are shown in Figure 1b. A remarkable difference was observed in the type-specific avidities elicited by GII.4 1999 and GII.4 2012 VLPs (Figure 1b). GII.4 1999 VLPs induced type-specific IgG antibodies with high avidity (mean avidity index 85.7 9%), whereas GII.4 2012 type-specific antibody avidity was poor (19.4 10.7%). As expected, the avidity of GII.4 1999 immune serum against heterologous VLPs was considerably lower than type-specific.