BioProject

Background: Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). More...

Background: Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. Methods: We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-timulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. Results: A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. Conclusions: We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Overall design: The study was performed in 2005. PBMCs were isolated from 2 healthy volunteers 13 days following vaccination with Vaccinia virus. Freshly isolated PBMCs were then stained with CD4, CD8, CD45RA and CD38 for cell sorting on a Becton Dickinson FACSAria. Lymphocytes were identified from an initial forward scatter-area vs side scatter-area plot, then single cells from side scatter-width vs side scatter-height and forward scatter-width vs forward scatter-height, a CD4 vs CD8 plot used to identify CD4 T cells and CD8 T cells. A CD38 vs CD45RA plot was drawn from both the CD4 and CD8 T cell populations. CD4 T cells were sorted into naïve (CD45RA+CD38dim), activated effector (CD45RA-negCD38+++) and resting memory (CD45RA-negCD38-neg). Activated CD8 T cells were sorted as CD38+++CD45RA+/neg. The sorted cell populations from the 2 vaccinated volunteers (S2 and S3) were assigned the following nomenclature S2P6 (activated effector CD4+), S2P7 (resting memory CD4+), S2P8 (naïve CD4+) and S2P9 (activated effector CD8+) for the first vaccinated volunteer and S3P7, S3P7, S3P8 and S3P9 for the second vaccinated volunteer. Less...