Simultaneous Immunization with Multivalent Norovirus VLPs Induces Better Protective Immune Responses to Norovirus than Sequential Immunization

Figures

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  Figure 1. Electron microscopy images of the purified norovirus (NoV) genotypes: (a) GI.3, (b) GII.4, (c) GII.17, and (d) GII.4 SYD virus-like particles (VLPs). VLPs were examined by FEI Tecnai F12 electron microscope (Philips 487 Electron Optics, Holland) after negative staining with 3% uranyl acetate, pH 4.6. Bar, 200 nm.

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  Figure 2. Immunization schemes of the study groups. Experimental groups of mice receiving monovalent (MV) (a) NoV VLPs (Gr I–IV) or the control group (Ctrl), receiving carrier only, were intramuscularly immunized twice at study weeks 0 and 3 and terminated at week 5. (b) Mice receiving a multivalent mix (MX, Gr V) of the four NoV VLPs and the RV VP6 protein was immunized using the same schedule but terminated at week 9. (c) Sequentially immunized mice (SQ, Gr VI) were primed twice at weeks 0 and 3 with the trivalent mix of NoV VLPs and RV VP6, boost immunized at weeks 5 and 7 with heterologous NoV VLPs, and terminated at week 9. Tail blood samples (◊) were collected at the indicated time points. Each horizontal arrow represents one injected antigen.

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  Figure 3. NoV genotype-specific IgG antibody responses induced by monovalent NoV VLPs or multivalent VLP mix immunization. Termination sera of mice immunized with monovalent (MV) VLPs, GI.3 (Gr I), GII.4-1999 (Gr II), GII.17 (Gr III), or GII.4 SYD (Gr IV), or with the multivalent NoV VLP mix (MX, Gr V), or the carrier only (Control group, Ctrl) were analyzed with enzyme-linked immunosorbent assay (ELISA). (a) The mean optical density (OD490) at the serum dilution 1:200 of individual mice is illustrated with group mean and the standard error of the mean (SEM). The horizontal dashed line indicates maximum background level (cut-off limit). Mean IgG end-point titration curves specific for NoV (b) GI.3, (c) GII.4, (d) GII.17, and (e) GII.4 SYD VLPs with the SEM are shown.

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  Figure 4. NoV genotype-specific IgG antibody responses induced by monovalent NoV VLPs or sequential VLP immunization. Termination sera of mice immunized with monovalent (MV) VLPs, GI.3 (Gr I), GII.4-1999 (Gr II), GII.17 (Gr III), or GII.4 SYD (Gr IV), or sequentially (SQ) immunized with heterologous VLP boosts (Gr VI) or the carrier only (Control group, Ctrl) were analyzed with enzyme-linked immunosorbent assay (ELISA). (a) The mean optical density (OD490) at the serum dilution 1:200 of individual mice is illustrated with the group mean and the standard error of the mean (SEM). The horizontal dashed line indicats maximum background level (cut-off limit). Mean IgG end-point titration curves specific for NoV (b) GI.3, (c) GII.4, (d) GII.17, and (e) GII.4 SYD VLPs with the SEM are shown.

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  Figure 5. NoV genotype-specific blocking antibodies after immunization with multivalent NoV VLP as a mix or sequentially. Individual sera of mice immunized with multivalent mix (MX, Gr V) or sequentially (SQ, Gr VI) were 2-fold diluted starting at 1:100 dilution and assayed for the blocking of homologous NoV (a) GI.3, (b) GII.4-1999, (c) GII.17, or (d) GII.4 SYD VLP binding to histo-blood group antigens present in pig gastric mucin. The blocking index (%) was calculated as [100% − [OD (wells with serum)/OD(wells without serum, maximum VLP binding)] × 100%] and shown are individual mouse 50% blocking titer (BT50) and group geometric mean titer with 95% confidence interval. An arbitrary titer, BT50 of 5, was assigned to samples with <50% blocking index at the lowest serum dilution 1:100. Statistical differences were determined using Fisher’s exact test, and a p value of ≤0.05 was considered statistically significant (*).

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  Figure 6. NoV cross-blocking antibodies after multivalent NoV VLP mix or sequential immunization. Group-wise pooled sera of mice immunized with multivalent mix (MX, Gr V) or sequentially (SQ, Gr VI) were 2-fold diluted starting at 1:20 dilution and assayed for the cross-blocking of heterologous NoV (a) GII.4-2006, (b) GII.4 NO, (c) GI.1, or (d) GI.4 VLP binding to histo-blood group antigens present in pig gastric mucin. The blocking index (%) was calculated as [100% − [OD (wells with serum)/OD(wells without serum, maximum VLP binding)] × 100%] and shown are group mean blocking indeces (%) with the standard errors of the mean of repeated assays. The horizontal dashed line represents a blocking titer of 50% (BT50).

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  Figure 7. Kinetics of NoV type-specific and cross-reactive blocking antibodies after multivalent NoV VLP mix immunization. Group-wise pooled tail blood samples of mice were collected at the time of the first immunization (week 0), the second immunization (week 3), and two and four weeks after the second immunization (weeks 5 and 7) and 2-fold diluted starting at 1:100 dilution, for assaying the blocking antibodies of (a) homologous GII.4 SYD VLPs and (b) heterologous GII.4 NO VLPs binding to histo-blood group antigens present in pig gastric mucin. The blocking index (%) was calculated as [100% − [OD (wells with serum)/OD(wells without serum, maximum VLP binding)] × 100%] and shown are titration curves of each time-point blocking index (%). The horizontal dashed line represents a blocking titer of 50% (BT50).

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  Figure 8. NoV cross-blocking antibodies after monovalent NoV GII.17 VLP immunization. Termination sera was pooled and serially 2-fold diluted starting at a 1:20 dilution and analyzed in a blocking assay against seven heterologous GI and GII VLPs. The blocking index (%) was calculated as [100% − [OD (wells with serum)/OD(wells without serum, maximum VLP binding)] × 100%] and shown are the blocking index (%) of the two analysis. The horizontal dashed line represents a blocking titer of 50% (BT50).