Detection of HIV Vaccine-Induced Cell-Mediated Immunity in HIV-Seronegative Clinical Trial Participants Using an Optimized and Validated Enzyme-Linked Immunospot Assay

Abstract

An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.

An HIV-1 clade A/E DNA prime, recombinant fowlpox virus boost vaccine is safe, but non-immunogenic in a randomized phase I/IIa trial in Thai volunteers at low risk of HIV infection

“…34,35 However, the STEP trial indicates that T-cell immunogenicity as judged by IFN-g/IL-2 responses does not imply protection. 26,27 Of note, current technology does not allow for the induction of potent neutralizing antibodies that may prove valuable in the search Responses are mean (sD). * p represents a formal comparison of change from baseline to week 13 for each antigen between vaccine and placebo arms using a Mann Whitney U test.…”

“…To be consistent with the STEP Study MRKAd5 HIV-1 Gag/Pol/Nef trivalent vaccine ELISpot data, the proportion of vaccinees exhibiting a positive response had to be greater than two thirds, i.e., four out of six volunteers in order to be deemed a positive result. 26 As there were only two volunteers in the control arm, mean responses were presented. The authors would like to thank Wadchara Pumpradit for his assistance in the preparation and initial drafting of trial protocols gag/pol, truncated env and modified tat/rev expressed under the control of the fowlpox early/late promoter.…”

Guidelines on good clinical laboratory practice: Bridging operations between research and clinical research laboratories

“…If non-FDA approved methods are utilized, such as to monitor immunogenicity to a candidate vaccine, the laboratory must define, test and document the parameters described in the ICH Guidelines, Validation of Analytical Procedures: Text and Methodology, Q2(R1) document that includes the original Q2A and Q2B documents [38], or the Bioanalytical Method Validation Guidelines provided for the Industry by the FDA [39] to validate a bioanalytical assay. Examples of bioanalytical assays that have been validated for use in human clinical trials, using the ICH Guidelines are the ELISpot [6,40] and ICS assays [7]. These include accuracy, precision, analytical sensitivity, analytical specificity, reportable range, reference intervals, and any other parameter required for test performance.…”

Retracing our STEP towards a successful CTL-based HIV-1 vaccine

“…To date, the interferon-γ ELISpot is the only validated assay for HIV-1 vaccine trials [8,9], by nature of its ease, reproducibility, and quantitative precision in mapping the targeting and magnitude of CTL responses. This assay provided the data that served as the basis for advancement of rAd5 into phase II human trials.…”