Bv780 cd45ra

Patient 355 (59-year old Male, infected with genotype 1a HCV, baseline viral load 467 000 IU/ml) received a prime vaccination of ChAd3-NSmut (2.5 × 10¹⁰ viral particles) and an MVA-NSmut (2 × 10⁸ plaque forming units) boost vaccination 8 weeks later. PBMC were collected 14 weeks post-boost vaccination for assessment of single cell gene expression (22 ). PBMC were thawed and prestained with a live/dead marker (Life Technologies, cat# L34976) and a PE-conjugated pentamer reagent (PE-labeled HCV NS31406–1415 (KLSALGINAV; HLA-A*0201)). The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 329505), PerCP-eFluor 710 LAG3 (eBioscience, cat# 46–2239), PE-Cy7 CCR7 (BioLegend, cat# 329919), APC CD39 (BioLegend, cat# 328209), BV421 PD1 (BioLegend, cat# 329919), BV510 CD3 (BioLegend, cat# 317332), BV605 CD8A (BioLegend, cat# 301040), BV780 CD45RA (BioLegend, cat# 304140).
The relevant institutional review boards approved all human subject protocols, and all subjects provided written consent before enrollment.

A healthy volunteer was vaccinated with a single dose (0.5 ml containing at least 10⁵ PFU) of 17D live-attenuated yellow fever vaccine strain administered subcutaneously. Seroconversion after vaccination was confirmed by assaying the neutralizing antibody titers for YF-17D (data not shown). A whole blood sample was obtained 9 months post-vaccination and lymphocytes were enriched from whole blood via Ficoll gradient centrifugation and a CD8 negative selection magnetic bead kit (Miltenyi Biotec). Cells were prestained with a live/dead marker (Life Technologies, cat# L34976) and an APC-labeled tetramer reagent (NS4B 214–222 LLWNGPMAV, kindly provided by Dr Rafi Ahmed). The cells were then stained with the following antibodies: FITC CD8A (BioLegend, cat# 300906), PE CXCR3 (BioLegend, cat# 353705), PE-Cy7 CCR7 (BioLegend, cat# 353226), BV421 IL2Rb (BioLegend, cat# 339009), BV510 CD3 (BioLegend, cat# 317332), BV605 CD95 (BioLegend, cat# 305627), BV780 CD45RA (BioLegend, cat# 304140).