We continued to investigate whether the advantages of three-component regimes could be achieved in a simplified two-stage regime, by mixing protein and adjuvant with one or both viral vector components (Fig. 4A and VX-770 clinical trial B). We found that there was no Modulators significant difference by Kruskal–Wallis test between the three-immunization regimes and a two-immunization regime mixing protein and Montanide ISA720 with both adenovirus prime and MVA boost. Interestingly, there was a small but statistically significant increase in CD8+ T cell responses and decrease in antibody responses with the (A+P)–M regime relative to A–P–M (P < 0.05, ANOVA with Bonferroni post-test).

Antibody responses tended to be highest with the three component regimes, or when protein-adjuvant was co-administered with both viral vectors. Interestingly, in

C57BL/6 mice, (A+P) priming induced modestly but significantly higher CD8+ T cell responses than adenovirus alone ( Fig. 1D, P = 0.04, Mann–Whitney test). Thus a simplified two-shot immunization regime appears highly immunogenic and mixing of the viral vectors with protein and adjuvant did not appear to affect vector potency, a result which may encourage development of further strategies combining vectors with protein and adjuvant, including homologous vector–protein prime–boost immunization regimes. Serum antibody and splenic T cell responses were assayed by ELISA and IFNγ ELISPOT 138 days after final vaccination for selected groups of mice (Fig. 2 D291 time point and Fig. 5). Antibody responses to A–M–P Anti-cancer Compound Library and A–P–M remained significantly higher than those for A–M (P < 0.05 for both comparisons by Kruskal–Wallis test with Dunn's multiple comparison post-test), while CD8+ T cell responses following A–M–P and A–M remained greater than those 3-mercaptopyruvate sulfurtransferase for A–P (P < 0.01 and P < 0.05 respectively by the same method). There was

a mean drop of 0.4 log units in ELISA titer between 14 and 138 days after final vaccination, with no significant difference in this rate of decline between groups ( Fig. 5C, P = 0.37 by Kruskal–Wallis test). Thus, as was the case with early post-vaccination responses, maximal long-lived IgG responses were detected with any regime including AdCh63 and protein, while any regime including AdCh63 and MVA induced maximal long-lived CD8+ T cell responses in the spleen. We also compared the antibody and CD8+ T cell responses of six mice receiving the A–M–P regime entirely intramuscularly versus six mice receiving the viral-vector components intradermally (i.d.) (Fig. 6). There was no significant difference by t-test between the two groups’ log ELISA titer (P = 0.26) or % IFNγ+ CD8+ T cells (P = 0.20) 14 days after final vaccination, nor was a difference found between groups for either ELISA or CD8+ T cell responses by repeat measures ANOVA taking into account all time points up to 14 days after final vaccination.