Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay.
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.
Dubey
Sheri
S
Department of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA. sheri_dubey@merck.com
Clair
James
J
Fu
Tong-Ming
TM
Guan
Liming
L
Long
Romnie
R
Mogg
Robin
R
Anderson
Kiersten
K
Collins
Kelly B
KB
Gaunt
Christine
C
Fernandez
V Rose
VR
Zhu
Lan
L
Kierstead
Lisa
L
Thaler
Scott
S
Gupta
Swati B
SB
Straus
Walter
W
Mehrotra
Devan
D
Tobery
Timothy W
TW
Casimiro
Danilo R
DR
Shiver
John W
JW
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
D016915:AIDS Vaccines; D015488:HIV Antigens; D010455:Peptides; D007371:Interferon-gamma