title: 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.
authors.affiliation: Department of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA. sheri_dubey@merck.com
authors.forename: Sheri
authors.initials: S
authors.lastname: Dubey
authors.forename: James
authors.initials: J
authors.lastname: Clair
authors.forename: Tong-Ming
authors.initials: TM
authors.lastname: Fu
authors.forename: Liming
authors.initials: L
authors.lastname: Guan
authors.forename: Romnie
authors.initials: R
authors.lastname: Long
authors.forename: Robin
authors.initials: R
authors.lastname: Mogg
authors.forename: Kiersten
authors.initials: K
authors.lastname: Anderson
authors.forename: Kelly B
authors.initials: KB
authors.lastname: Collins
authors.forename: Christine
authors.initials: C
authors.lastname: Gaunt
authors.forename: V Rose
authors.initials: VR
authors.lastname: Fernandez
authors.forename: Lan
authors.initials: L
authors.lastname: Zhu
authors.forename: Lisa
authors.initials: L
authors.lastname: Kierstead
authors.forename: Scott
authors.initials: S
authors.lastname: Thaler
authors.forename: Swati B
authors.initials: SB
authors.lastname: Gupta
authors.forename: Walter
authors.initials: W
authors.lastname: Straus
authors.forename: Devan
authors.initials: D
authors.lastname: Mehrotra
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Tobery
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.forename: John W
authors.initials: JW
authors.lastname: Shiver
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology* / Q000627:therapeutic use; D002986:Clinical Trials as Topic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005189:False Positive Reactions; D015488:HIV Antigens / Q000032:analysis* / Q000276:immunology; D015658:HIV Infections / Q000517:prevention & control; D018023:HIV Seronegativity; D015497:HIV-1; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010455:Peptides / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D016915:AIDS Vaccines; D015488:HIV Antigens; D010455:Peptides; D007371:Interferon-gamma