Inhibition of T cell activation by CPXV 219 is species-specific.

<p><b>A)</b> Human <i>Mtb</i>-specific T cell clone D466 D6 was incubated with BEAS-2b cells uninfected (UN) or infected with VACV or VACV-219 (3 h) prior to addition of CFP10_2-12 peptide. The number of IFNγ+ T cells was determined by ELISPOT. <b>B)</b> CM9-specific T-cells were incubated with HFF infected with VACV or VACV-219 for 18 h and stimulated with CM9-peptide pulsed BLCLs. The percentage of INFγ+ TNFα+ CD8+ T cells was determined by ICCS. <b>C)</b> PBMC from VACV-immune subjects (n = 3) were infected with indicated viruses (optimized MOI of 0.3-0.6) for 18 h. The infection rates for CD14+ cells were VACV (54%), CPXV (45%), CPXV Δ12Δ203-221 (51%), CPXVΔ12-203 (60%), CPXVΔ11-38 (72%), and CPXVΔ204-221 (73%). The percentage of CD4+ and CD8+ responding to poxvirus infection was determined by ICCS for IFNγ and TNFα. The frequency of VACV-reactive T cells was set to 100%. <b>D)</b> Splenocytes from VACV-immunized mice were incubated with A20 cells infected with indicated viruses (MOI 5.0) for 6 h. The frequency of poxvirus-reactive T cells was determined by ICCS for IFNγ and TNFα relative to the frequency of VACV-reactive T cells which was set to 100%. <b>E)</b> Biosynthesis of CPXV219 was studied in human 143 cells infected with CPXV wild-type or Δ219 mutant (3 h) and metabolically labeled for 45 min followed by chase for 0.5, 1, and 3 h. Cell lysates were immunoprecipitated with αCPXV219 Ab. <b>F)</b> Left Panel: Immunoblot of CHO cells infected with CPXV, CPXVΔ219 (MOI = 5.0) or uninfected (UN) using αCPXV219 Ab. Right Panel: Immunoblot with αCPXV219 Ab of CHO cells infected with VACV, VACV-219 (MOI = 5.0) or uninfected (UN), or co-infected with T7-polymerase expressing VACV VTF7-3 (MOI = 5.0).