Quantitative analysis of cell-mediated immune responses induced by candidate HIV vaccines requires robust procedures for collecting and processing human peripheral mononuclear blood cells (PBMCs). We evaluated several parameters in order to optimize a sample handling process that would be suitable for a multicenter clinical trial. Among the findings, systematic increases in the magnitude of IFN-gamma ELISpot responses were observed when the time from blood collection to PBMC freezing was reduced to <12 h. By implementing these improvements within an ongoing clinical trial, the estimated immunologic response rates to an adenovirus- based HIV vaccine increased by more than 20 percentage points to approximately 80% of the vaccine recipients against any of the vaccine antigens and the average levels of T cell response improved more than 3-fold. These studies establish the importance of optimal conditions for PBMC collection and handling to the success of a clinical development program.

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.

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Aedes (Stegomyia) cretinus is a rarely documented mosquito with a Mediterranean distribution, whereas Aedes (S.) albopictus has spread worldwide in the past two decades because of its anthropogenic associations. A third closely related species, Aedes (S.) flavopictus, is sympatric with A. albopictus in northeast Asia. The three species are characterized by a striking mid-thoracic white stripe and, consequently, field-collected individuals may be difficult to separate by morphology. Sixteen biochemical markers were described for laboratory strains representing the three species; these provided the first biochemical genetic profile for A. cretinus and A. flavopictus. Diagnostic enzymes for identifying each species pair were determined. A biochemical key was provided to distinguish among adults of the three species. Several enzyme loci that were diagnostic for the adult stage proved unreliable for identifying immature stages. Voucher specimens for link-reared series of larva, pupa, adult male, and adult female stages of the A. cretinus Crete strain (n = 88) and the A. albopictus Nepal strain (n = 105) were deposited at the Yale Peabody Museum of Natural History, New Haven, CT.