The response to DNA immunization with Env was examined initially for BALB/c mice by mapping epitopes that were recognized by T cells. Vaccination with a plasmid DNA encoding a modified clade B Env immunogen revealed a CD4 response to four peptide pairs that showed reactivity above the threshold level of 0.1%, a >5-fold increase above the background levels with control peptides, by ICS for IFN-γ and TNF-α (Fig. , upper panel). In contrast, ADV vector immunization revealed a response to two epitope pairs, only one of which was the same as that stimulated by plasmid DNA vaccination (Fig. , middle panel). In contrast, DNA/ADV prime-boost vaccination induced a response to at least 10 epitope pairs (Fig. , lower panel). All the responses induced by DNA immunization were present within this pool, whereas only one of the adenoviral immunization epitope pairs was detected. The DNA/ADV immunization therefore elicited a stronger immune response to at least five epitopes that had not been detected with immunization by either DNA or ADV alone. In contrast, mapping of the CD8 epitopes revealed a different pattern of epitope responses. Two immunodominant peptide pairs in the V3 region were identified, and these epitopes were recognized comparably by CD8 cells from animals immunized with DNA and ADV alone (Fig. , upper and middle panels). While DNA/ADV immunization increased this response nearly fivefold, the same epitopes were recognized, suggesting a quantitative effect of the boost CD8 epitope recognition (Fig. ).
FIG. 1. Definition of CD4 and CD8 epitope responses to HIV Env in BALB/c mice. BALB/c mice were injected with DNA, ADV, or DNA/ADV vectors encoding Env, as indicated in Materials and Methods, and ICS to peptide pairs in the indicated positions was determined. (more ...)
To evaluate whether this response was independent of the mouse strain and therefore generalizable, the CD4 and CD8 responses of C57BL/6 mice to the Env epitopes were mapped. In this strain, fewer CD4 epitopes were elicited by the ADV immunization than by the DNA alone (Fig. , middle panel versus upper panel). Again, the DNA/ADV prime-boost immunization stimulated a response to several different epitope pairs compared to the DNA vaccine alone (Fig. , lower panel versus upper panel). The CD8 response to Env in BALB/c mice was difficult to evaluate because of the absence of a substantial response to any epitope in this strain (Fig. ). Only two responses induced by ADV alone were observed, of marginal significance, and no substantial effects were seen using DNA or DNA/ADV vaccination (Fig. , middle panel versus upper and lower panels), suggesting that this strain is a nonresponder for the Env antigen.
FIG. 2. Analysis of CD4 and CD8 epitope responses of C57BL/6 mice to HIV Env. C57BL/6 mice were injected with DNA, ADV, or DNA/ADV vaccines for Env, as indicated in Materials and Methods, and ICS to peptide pairs in the indicated positions was determined. The (more ...)
FIG. 3. Characterization of CD4 and CD8 epitope responses of BALB/c mice to HIV Gag. BALB/c mice were injected with the indicated vectors encoding Gag as described in Materials and Methods, and ICS to the indicated pairs of peptides was determined. The peptides (more ...)
The immune responses to alternative immunogens were next analyzed to determine whether diversification of the CD4 response seen with DNA/ADV vaccination applied to an independent antigen, Gag. For BALB/c mice, the finding observed was similar to that for the Env response. Only two epitope pairs were recognized, slightly above background levels, after DNA immunization (Fig. , upper panel). A larger number of pairs (15) were observed after adenoviral immunization alone, but the magnitude and diversity of the response was greater with the DNA/ADV boost, where responses to 20 pairs were detected by ICS of CD4 cells (Fig. , middle panel versus lower panel). Of these epitopes, the majority differed from those stimulated by either DNA or ADV boost alone. In contrast, six epitope pairs were identified with the CD8 response to Gag in BALB/c mice after DNA vaccination, and an additional one was seen after ADV priming alone (Fig. , upper panel versus lower panel). Similar to the Env response, CD8 ICS increased in magnitude but retained the same specificity of epitope response as DNA or ADV vaccination alone (Fig. , lower panel versus middle and upper panels).
Finally, the response to Gag in C57BL/6 mice was analyzed. While three responses were observed above background for DNA alone and six for ADV alone, eight were observed with DNA priming and ADV boosting, of which four had not been seen with either agent alone (Fig. , lower panel versus middle and upper panels). In contrast, the CD8 response to Gag in C57BL/6 mice was focused on three epitope pairs, all of which were identical and boosted in magnitude with the DNA/ADV immunization (Fig. , lower panel versus middle and upper panels).
FIG. 4. Specificities of the CD4 and CD8 epitope responses of C57BL/6 mice to HIV Gag. C57BL/6 mice were immunized with the specified Gag immunogen vectors as described in Materials and Methods, and ICS to peptide pools from the HIV Gag region was determined. (more ...)