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

Abstract

The gamma interferon (IFN-␥) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In orderto 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-␥ 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-␥ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-␥ 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.Most human immunodeficiency virus (HIV) vaccines currently in development aim to induce cellular immune responses, since these have been shown previously to temporally correlate with the containment of virus in infected individuals and, more significantly, to be crucial in the suppression of virus in the rhesus macaque model (2,13,15,25). The ability to measure and quantitate cellular immune responses has been facilitated through the development of enzyme-linked immunospot (ELISPOT) and flow cytometry assays which determine the number of antigen-specific cells through surrogate markers of effector function, such as cytokine production or the degranulation of lytic granules (1,8,23,29), and are more quantitative and less labor-intensive than traditional assays that detect T-cell responses, such as 51 Cr release and lymphoproliferation assays (19). The gamma interferon (IFN-␥) ELISPOT assay is a primary assay employed to measure vaccine immunogenicity in HIV vaccine clinical trials, in addition to trials in the cancer, malaria, and tuberculosi...

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

“…Studies of the effects of freezing/thawing on PBMCs have been performed with blood of T1D patients [35,44], healthy subjects and patients with other diseases such as HIV infection [24,45,46]. As expected, all show that fresh blood yields more viable cells than frozen PBMCs.…”

Safety and Immunogenicity of DNA Prime and Modified Vaccinia Ankara Virus-HIV Subtype C Vaccine Boost in Healthy Adults

“…For all other assays, PBMC were frozen in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) using a Kryo 560-16 rate controlled freezer (Planer, Sunbury-On-Thames, United Kingdom) and stored in vapor phase liquid nitrogen. The PBMC for the IFN-␥ ELISPOT assay were plated at 2 ϫ 10 5 viable cells per well in quadruplicate with peptides at 1.5 g/ml, representing the vaccine inserts, as described previously (39)(40)(41). Peptides were synthesized as 15-mers overlapping by 11 amino acids with ϳ90% purity by high-pressure liquid chromatography (HPLC) (AnaSpec Inc., Fremont, CA).…”

“…A peptide pool consisting of 9-or 10-mer peptides representing cytomegalovirus, Epstein-Barr virus, and influenza virus (CEF) was used at 1.5 g/ml, phytohemagglutinin (PHA) was used at 10 g/ml, and a mock stimulus (DMSO-medium) was used as previously described (40,42).…”

Quality Monitoring of HIV-1-Infected and Uninfected Peripheral Blood Mononuclear Cell Samples in a Resource-Limited Setting

“…Many of the T-cell-based assays used to assess potential HIV-1 candidate vaccines have been standardized and evaluated to characterize vaccine immunogenicity (7,22,43) but may not define correlates of protection (8,35). T cells are linked to control of HIV-1 infection, and specific functional profiles are associated with slow HIV-1 disease progression in infected individuals (6).…”

“…Analytic variables that impact PBMC processing include separation method, type of cryoprotectant medium, prechilling of the freeze containers, cryopreservation parameters, and temperature of the thawing medium (9,15,28,30,37). In addition to defining processing parameters, it is important to monitor quality assurance for cryopreserved PBMC assays after cryopreservation (7). However, few studies have addressed the importance of continuous monitoring of the processing and cryopreservation of PBMC themselves but have focused instead on the procedures for thawing of cells in labs that perform end-point assays.…”