A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X, S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance
Figure 2
Ex vivo lymphocyte proliferation assays of T cells from GS-4774-immunized mice.
(A) Proliferation of inguinal LN cells harvested from GS-4774- or Yvec-immunized BALB/c mice. LN cells from 5 immunized mice were pooled and placed into in vitro stimulation with the indicated antigens for 4 days, followed by a 20 h 3H-thymidine uptake assay. In vitro stimulants are: Pichia pastoris yeast expressed, purified HBsAg and HBcAg, 3 µg/mL each; IPQSLDSWWTSL (HBsAg Ld restricted peptide) and AYRPPNAPI (HBcAg Ld restricted peptide), 10 µg/mL each; recombinant E. coli- expressed full length HBxAg, 3 µg/mL. P values, GS-4774 vs. Yvec: yeast expressed HBsAg & HBcAg, 0.0001; IPQSLDSWWTSL & AYRPPNAPI peptides, 0.089; recombinant HBxAg, 0.002. (B) Proliferation of CD4+ T cells isolated from splenocytes of GS-4774- or Yvec-immunized BALB/c mice. MACS-isolated splenic CD4+ T cells were stimulated with the indicated HBV antigens and assayed as in (A). Yeast expressed HBsAg & HBcAg stimulants: same as for panel A; GYHGSSLY, a MHC class II HBsAg mimetic peptide plus WGPSLYSIL, a 2-Dd restricted HBsAg peptide (10 µg/mL each). P values, GS-4774 vs. Yvec: yeast expressed HBsAg & HBcAg, 0.034; GYHGSSLY and WGPSLYSIL, 0.023. 3H-thymidine uptake is reported with medium background subtracted counts per minute (cpm) for each antigen stimulation condition. Error bars: standard error (s.e.) for replicate stimulations of the pooled immune cells.