3.5. ORF8-specific IgG subclasses and IgM
The five groups all elicited apparent ORF8-specific Th2-type IgG1 (Fig. 3B) and Th1-type IgG2a titers (Fig. 3C). The ORF8-PEG group showed the highest ORF8-specific IgG1 and IgG2a titers in the four groups on day 21. In particular, the Th1-type ORF8-specific IgG2a titer of the ORF8-PEG group was 1.1×104 at the third dose, which was equal to the ORF8-specific IgG1 titer (1.1×104). This indicated that self-conjugation with 8-arm PEG induced strong Th1-type and Th2-type immune response to ORF8. Moreover, the IgG1 titers of the ORF8-CRM group at the third dose (6.0×103) were approximately equal to that of the ORF8/AL group (6.2×103). The IgG2a titers of the ORF8-CRM group at the third dose (1.8×103) were lower that of the ORF8/AL group (5.1×103). The IgG2a/IgG1 of the ORF8 group was 0.84, whereas the IgG2a/IgG1 values of the ORF8-CRM, ORF8-PEG and ORF8/AL groups was 0.30, 1.00 and 0.82, respectively. Thus, conjugation with CRM197 and self-conjugation with 8-arm PEG both altered the Th1/Th2 bias.
As shown in Fig. 3D, the ORF8 group showed very low ORF8-specific IgM titers (4.8×102). The ORF8-CRM group (1.4×103) showed higher IgM titers than the ORF8 group (P<0.05). The IgM titer of the ORF8-PEG group (3.6×103) was higher than that of the ORF8 group (P<0.05) and comparable to that of the ORF8/AL group (3.7×103). Because the IgG/IgM ratio of the ORF8-PEG group was higher than that of the ORF8/AL group, this suggested that self-conjugation with 8-arm PEG shifted more immune response from IgM to IgG than the ORF8/AL group. This was typical for the protein-based vaccines.