FIG 4.

First study of specific (tgD) immune responses in mice immunized with aBNBD3/pMASIA-tgD complexed vaccines. C57BL/6 mice (n = 8) were immunized twice intradermally with 5 μg pMASIA or pMASIA-tgD complexed with either 0 nmol, 0.01875 nmol (low), 0.1875 nmol (medium), or 1.8750 nmol (high) of aBNBD3 (analog in which glycine replaces glutamine in positions 1 and 27). (a to c) tgD-specific IgG1 titers 15 days after the second immunization (a) and IgG2a (b) and IgG (c) titers 1 month after the second immunization were determined by ELISA. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a reading 2 standard deviations above the negative control. (d) One month after the second immunization, virus-neutralizing antibodies in serum were measured. VN titers are expressed as the reciprocal of the highest dilution of serum that resulted in <50% of cells displaying cytopathic effects. (e and f) The numbers of tgD-specific IFN-γ-secreting (e) and IL-5-secreting (f) splenic cells were measured by ELIspot assay. ELIspot results are expressed as the difference between the numbers of IFN-γ- or IL-5-secreting splenic cells in tgD-stimulated wells and medium-control wells per 10⁶ cells. Bars represent the median values for each group, with interquartile ranges. Significant differences between groups are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.