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Detection of Merck Ad5 Vaccine Vector and HIV-1 Insert Genes in HVTN 071: a Trial Using the MRKAd5 gag/ pol/nef Vaccine from the Step Study. Tuofu Zhu, MD, PhD // Haiying Zhu, MS Department of Laboratory Medicine University of Washington . HVTN 071 Trial.
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Detection of Merck Ad5 Vaccine Vector and HIV-1 Insert Genes in HVTN 071: a Trial Using the MRKAd5 gag/pol/nef Vaccine from the Step Study
Tuofu Zhu, MD, PhD // Haiying Zhu, MS
Department of Laboratory Medicine
University of Washington
1.5 x 1010 genomes
IM inoculation (n=35)
To develop a new ultrasensitive PCR assay capable of detecting extremely low levels of Merck Ad5 vector, HIV-1 inserts (gag, pol and nef) and wild type Ad5 undetectable by traditional assay.
To determine the presence and persistence of the Merck Ad5 vector, HIV-1 inserts (gag, pol and nef) and wild type Ad5 in the PBMC of vaccinated subjects.
The Zhu lab previously used a sensitive limiting-dilution nested PCR to detect HIV-1 proviral DNA from two seronegative subjects (Zhu et al., J. Virol. 77: 6108-6116, 2003) and low levels of SIV proviral DNA in macaques (Zhu et al., Virology 323:208-209, 2004).
The Zhu lab has recently developed a supersensitive PCR assay that meets the requirements for large scale screening of extraordinarily low levels of HIV-1 infection. This new PCR method can reliably detect a single HIV-1 template, which may be undetectable by traditional real time and nested PCR methods.
Based on HIV-1 detection platform, we designed specific primers and probes for Merck Ad5 vaccine construct, HIV-1 insert genes and wild type Ad5 to detect their presence and persistence in the vaccinated subjects
Detection of HIV-1 provirirus from patients genomic DNA contaning 50 or less HIV-1 copies/million celss
2nd round PCR utilizing Real Time PCR system
Screening for positives
Sequence the amplified Ad5 vaccine construct and HIV-1 inserts
Calculate the copy numbers
HIV-1 gag/Vaccine Ad5
HIV-1 pol/Vaccine Ad5
HIV-1 nef/Vaccine Ad5
WT Ad5/Vaccine Ad5
Detection rates were calculated as the number of positive per total PCR performed.
Assuming perfect conditions of sensitivity and specificity, the probability of obtaining a positive reaction from a dilution of 1 copy/reaction is 63% (Hughes, J.P, et al., 59:505-511, Biometrics 2003).
Tuofu Zhu Lab
Dominic M. Forte
Molecular Diagnostic lab
HVTN Laboratory Program
Lynne A. Becker
Andrew J. Bett
Danilo R. Casimiro