Measuring T-cell immunity in HIV vaccine clinical trials: PBMC and ELISPOT assay proficiency concordance in laboratories from 3 continents
Abstract
The interferon-gamma (IFN-{gamma}) ELISPOT assay is used routinely to evaluate the potency of HIV and other vaccine candidates. In order to compare candidates and pool data across multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intra-laboratory as well as assay performance over time. Seven IAVI-sponsored trial sites participated in the proficiency panels. At each laboratory two operators independently processed identical panels containing frozen peripheral blood mononuclear cell (PBMC) samples from different donors using four blinded stimuli. PBMC recovery and viability after overnight rest and IFN-{gamma} ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting: median recovery (78%) and viability (95%). The laboratories were able to detect similar antigen-specific T cell responses ranging from 50 to >3000 spot forming cells per million PBMC. An approximate range of a half log across operators within or across sites was seen when comparing antigen specific responses. Consistently low background responses were seen in all laboratories. This proficiency panel demonstrates the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-{gamma} ELISPOT responses. This panel also illustrates the ability to standardize the IFN-{gamma} ELISPOT assay across multiple laboratories when common training, reagents such as FCS and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV and other vaccines.
Citation
Anzala, O.; Birungi, J.; Boaz, M.J.; Cooper, A.; Cox, J.H.; Dally, L.; Farah, B.; Gill, D.K.; Gilmour, J.; Goonetilleke, N.; Hayes, P.; Indangasi, J.; Kaleebu, P.; Kitandwe, P.; Mahalingam, J.; Mahmoud, A.; Mhlanga, P.; Mishra, S.; Moore, S.; Narayanan, P.R.; Ogola, S.; Pujari, A.; Ramanathan, V.D.; Sathyamoorthy, P.; Seamons, L.; Semaganda, A.; Stevens, G.; Tarragona, T.; Thakar, M.; Van Eeden, M. Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents. Clinical and Vaccine Immunology (2009) 16 (2) 147-155. [DOI: 10.1128/CVI.00326-08]