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. 2011 Oct;79(10):4260–4275. doi: 10.1128/IAI.05214-11

Fig. 7.

Fig. 7.

Essentiality of each component of the tricomponent complex and effects of the ligand arrangement on the immune response. Mice were immunized by the subcutaneous or intranasal route three times, at weeks 0, 2, and 4, and antisera were collected 2 weeks after the third immunization in order to evaluate the Pvs25-specific IgG titers. All mice received 30 μg of the Pvs25H-A antigen as a conjugated or unconjugated protein. Cholera toxin (CT) (1 μg) was used as the intranasal adjuvant. Antibody titers were defined as described in the legend to Fig. 4. Asterisks indicate significant differences from the unimmunized control group by the Wilcoxon-Mann-Whitney test (*, P < 0.05), among the four groups indicated by the Kruskal-Wallis test (**, P < 0.001), or between the two groups indicated by the Wilcoxon-Mann-Whitney test (***, P < 0.01). (a) Female BALB/c mice (seven or four per group) were immunized with one of the following, from left to right: the Pvs25H-A antigen alone (30 μg), the COMP-spacer:Pvs25H-A fusion complex (36.3 μg), the antigen fused directly to the monomeric Z domain (34.5 μg), the COMP-Z:Pvs25H-A tricomponent complex (40.8 μg), or a mixture of the antigen (30 μg), the COMP-spacer (6.3 μg), and the Z domain (4.5 μg). The data for four groups (the antigen alone, the antigen mixed with CT, and the tricomponent complex administered by the s.c. or i.n. route) are duplicates of the data presented in Fig. 4b. (b) Female BALB/c mice (seven or four per group) were immunized with the ZV:Pvs25H-A fusion complex (43.2 μg) or the COMP-Z:Pvs25H-A tricomponent complex (40.8 μg). The ZV:Pvs25H-A fusion complex was generated by reacting a free sulfhydryl group of the C-terminally introduced Cys residue in the ZV delivery molecule, which consists of five tandemly repeated Z domains, with the SPDP-modified Pvs25H-A antigen. Chemical conjugation between the antigen and the ZV delivery molecule was confirmed by a human IgG-ELISA using an anti-Pvs25 antiserum (left) prior to immunization experiments (right).