P24.09
Background: The ELIspot assay has been used both for answering basic research questions and for immunogenicity assessments of HIV-1 vaccine candidates in clinical trials. As part of the quality management systems, IAVI-sponsored vaccine trial laboratories participate in monthly external quality assurance (EQA) proficiency panels that assess and monitor laboratory performance in ELIspot over time.
Methods: Frozen PBMC samples isolated from HIV-1 seronegative individuals with previously-characterized IFN-γ ELISpot responses to CMVpp65, FEC (Flu, EBV and CMV) and mock peptide pools were provided by IAVI from blood packs obtained from the South African National Blood Transfusion Service. Sufficient vials were provided to test the same 6 PBMC samples per month for 6 months. Two such PBMC panels were provided each year. Monthly testing was rotated amongst three laboratory staff at KAVI-Institute of Clinical Research (ICR). Cell viabilities and recoveries were also analyzed.
Results: Mock data were less than 50 SFU per million PBMC for all sets of PBMC tested over 12 months. 213 out of a total of 216 data points (98.6%) for mock, FEC and CMVpp65 responses were in the expected ranges for PBMC samples tested over 12 months. Intra-operator analysis showed that there were no statistically significant differences in the mock (p=0.35), FEC (p=0.99) and CMVpp65 (p=0.99) responses for PBMC tested over 12 months. Cell recovery was in the range of 3.0-13.1×106/ml with viability of above 92%.
Conclusions: Three operators have demonstrated competence in ELIspot testing of multiple batches of frozen PBMC over 12 months. Responses were within the expected ranges for mock, CMVpp65 and FEC among different operators. The Elispot proficiency therefore remains a robust and reproducible tool for the assessment of immunogenicity of HIV-1 and other vaccine candidates in clinical trials.
