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Clinical and Vaccine Immunology, February 2009, p. 147-155, Vol. 16, No. 2
1071-412X/09/$08.00+0     doi:10.1128/CVI.00326-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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 {triangledown}

Mark J. Boaz ,1,{dagger},{ddagger} Peter Hayes,2,{dagger} Tony Tarragona,2 Laura Seamons,2 Andrew Cooper,2 Josephine Birungi,3 Paul Kitandwe,3 Aloysius Semaganda,3 Pontiano Kaleebu,3 Gwynneth Stevens,1 Omu Anzala,4 Bashir Farah,4 Simon Ogola,4 Jackton Indangasi,4 Patrick Mhlanga,5 Melanie Van Eeden,5 Madhuri Thakar,6 Ashwini Pujari,6 Shadri Mishra,6 Nilu Goonetilleke,7 Stephen Moore,7 Abdul Mahmoud,7 Pattabiraman Sathyamoorthy,8 Jayashri Mahalingam,8 Paranji R. Narayanan,8 Vadakkuppattu D. Ramanathan,8 Josephine H. Cox,1* Len Dally,9 Dilbinder K. Gill,2 and Jill Gilmour2

International AIDS Vaccine Initiative, New York, New York,1 International AIDS Vaccine Initiative Core Laboratory, Imperial College, London, United Kingdom,2 Uganda Virus Research Institute, Entebbe, Uganda,3 Kenya Aids Vaccine Initiative, Nairobi, Kenya,4 Contract Laboratory Services, Johannesburg, South Africa,5 National Aids Research Institute, Pune, India,6 Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom,7 Tuberculosis Research Centre, Chennai, India,8 EMMES Corporation, Rockville, Maryland9

Received 8 September 2008/ Returned for modification 9 October 2008/ Accepted 9 December 2008

The gamma interferon (IFN-{gamma}) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from 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 intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-{gamma} ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate 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. These findings also illustrate the ability to standardize the IFN-{gamma} ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.

* Corresponding author. Mailing address: IAVI, 1600 Gude Dr., Rockville, MD 20850. Phone: (301) 251-8360. Fax: (301) 424-3120. E-mail: jcox{at}iavi.org

{triangledown} Published ahead of print on 17 December 2008.

{dagger} M.J.B. and P.H. contributed equally to the work.

{ddagger} Present address: Sanofi Pasteur, Swiftwater, PA.

Clinical and Vaccine Immunology, February 2009, p. 147-155, Vol. 16, No. 2
1071-412X/09/$08.00+0     doi:10.1128/CVI.00326-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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