Importantly, similar patterns to those previously observed were apparent from the lower dose experiment.

As expected all antibody and T GSK1349572 cell line cell responses were substantially weaker when using lower vaccine doses. Responses to protein–protein vaccination were markedly more variable than responses to adenovirus-containing regimes. At these lower doses, addition of protein did not enhance the antibody immunogenicity of viral vector regimes, with no significant differences in ELISA titers following A–M, A–P, A–M–P or A–P–M vaccination. T cell responses were again substantially higher in the A–M, A–M–P and A–P–M groups than in the A–P group. As before, the (A+P)–M, A–(M+P) and (A+P)–(M+P) two-stage regimes mixing viral and protein vaccines produced results DAPT in vivo similar to three-stage vaccination, with a trend towards higher antibody but lower CD8+ T cell responses in the group receiving (A+P)–(M+P). Thus despite the clearly sub-maximal responses achieved in these animals (in particular with the protein only vaccination), regimes

incorporating adenovirus and MVA again appeared to result in more consistent combined antibody and CD8+ T cell responses to the antigen. To further characterize the immune responses to the various vaccine modalities, we performed IgG isotype ELISAs. It was not possible to measure isotype-specific titers for the three P–P immunized mice with low total IgG ELISA titers. Bearing in mind this limitation, viral-vector-containing regimes induced a significantly greater ratio of IgG2a to IgG1 than was present in the high-total-titer P–P immunized mice, and that the IgG2a/IgG1 ratio was higher for all groups

137 days rather than 14 days after the final vaccination, corresponding to better maintenance of the titer of IgG2a than IgG1 over time (Fig. 7; P < 0.001 for both comparisons by repeated measures two-way ANOVA with Bonferroni's post-test). There was no interaction of out time and regime (i.e. no inter-regime differences in the rate of change of the IgG isotype balance over time). We continued to investigate the responses to the various regimes by measuring antibody avidity using NaSCN antibody-displacement ELISA for selected groups and time points (Fig. 8A–C). Among mice receiving A–M and A–P regimes, we observed that mice receiving A–M had higher antibody avidity 14 days post-boost than those receiving A–P, without any significant difference between 57 day and 97 day dose interval (Fig. 8A; P = 0.024 for regime comparison, P = 0.33 for comparison dose interval by two-way ANOVA).