Figure 4. Immunogenicity of rMVA-pp65-IGR3 in HHDII transgenic mice.

(A) HLA-A2 pp65-specific IFN-γ⁺ T cell responses generated by CD8⁺ splenocytes from transgenic HHDII mice immunized with purified rMVA-pp65-IGR3. HHDII mice (n=4) were inoculated intraperitoneally (i.p.) with 5×10⁷ pfu of rMVA-pp65-IGR3. Splenocytes were harvested 3 weeks post-immunization and assessed for IFN-γ production ex-vivo and after in-vitro stimulation (IVS) to a known HLA-A2-restricted pp65 epitope (pp65_495–503) by FACS analysis. Shown is a representative mouse for the ex-vivo and IVS groups. Mock groups represent re-stimulation with irrelevant peptide. (B) pp65_495–503-specific IFN-γ⁺ responses generated ex-vivo by CD8⁺ splenocytes from HHDII mice (n=4) immunized i.p. with 5×10⁷ pfu of purified rMVA-pp65-IGR3. Error bars represent SEM. Differences in pp65_495–503-specific IFN-γ⁺ responses compared to mock stimulated responses were evaluated using the Mann-Whitney test and a P value < 0.05 is considered significant. Details of the approaches can be found in Materials and Methods. (C) pp65_495–503-specific IFN-γ⁺ responses generated ex-vivo by CD8⁺ splenocytes from HHDII mice (n=4) immunized i.p. with 5×10⁷ pfu of either purified rMVA-pp65-IGR3 or rMVA-pp65-del II. Error bars represent SEM. Differences in responses were evaluated using the Mann-Whitney test and a P value < 0.05 is considered significant. (D) pp65_495–503-specific IFN-γ⁺ responses generated through IVS by CD8⁺ splenocytes from HHDII mice (n=4) immunized i.p. with 5×10⁷ pfu of purified rMVA-pp65-IGR3 after 7 passages were compared to responses from mice immunized i.p. with 5×10⁷ pfu unpassaged rMVA-pp65-IGR3. Error bars represent SEM. Differences in responses were evaluated using the Mann-Whitney test and a P value > 0.05 is not considered significant.