Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories

Abstract

BackgroundThe Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.MethodsWe describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.FindingsDetection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.InterpretationThe described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.

Human Leukocyte Antigen Variants B*44 and B*57 Are Consistently Favorable during Two Distinct Phases of Primary HIV-1 Infection in Sub-Saharan Africans with Several Viral Subtypes

“…In other populations, B*44:02 is a major allele that is often found on the B*44-C*05 haplotype (53). Understanding the early immune responses facilitated by various B*44 alleles should help with the dissection of protective immunity to HIV-1 infection, especially in the context of subtypes A1 and C in sub-Saharan Africa (22). Viral subtypes are highly relevant to the design of vaccines (42,57), and multiple reports have indicated that disease progression may differ somewhat by viral subtype (32,33,40,62,90).…”

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).…”

Statistical methods for the assessment of EQAPOL proficiency testing: ELISpot, Luminex, and Flow Cytometry

“…There have been earlier attempts at evaluating ELISpot proficiency panels and assay equivalence (Cox et al, 2005; Boaz et al, 2009; Gill et al, 2010), which focused primarily on positive or negative responses and concordance among labs. The EQAPOL ELISpot program does not have criteria for defining positive or negative responses, rather the goal is to assess how accurate to the consensus average and precise laboratories are using their own in-house assay given standard peptides and samples.…”