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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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tuofu zhu md phd haiying zhu ms department of laboratory medicine university of washington

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

slide2

HVTN 071 Trial

  • HVTN 071 was a trial administering MRKAd5 gag/pol/nef vaccine, designed to testthe safety and immune response of the vaccine used in the Step trial.
  • 35 subjects were vaccinated, who had varying pre-vaccination immunity to the vaccine vector (wild type Ad5)
  • The failure of the Step Study raised many questions, and to further define the mechanism of the vaccine, we utilized samples from the HVTN 071 trial to answer the following questions:
    • Do the Ad5 vaccine vector and HIV-1 insert genes persist in humans post vaccination?
    • If the vector persists, then how do vector-harboring cells interact with the immune system?
hvtn 071 vaccination schedule
HVTN 071 Vaccination Schedule

1.5 x 1010 genomes

MrkAd5 gag/pol/nef

IM inoculation (n=35)

Dose 1

(n=35)

Dose 2

(n=24)

Day 0

Day 28

Weeks 15-22

Week 31

Weeks 52-56

PBMC or

Leukapheresis

(n=8)

PBMC or

Leukapheresis

(n=8)

PBMC or

Leukapheresis

(n=8)

PBMC or

Leukapheresis

(n=1)

slide4

Objectives

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.

slide5

Assay Background

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

slide6

One Copy Detection of Extraordinarily Low Levels of HIV-1 Infection

Detection of HIV-1 provirirus from patients genomic DNA contaning 50 or less HIV-1 copies/million celss

slide7

Assay for Detecting Ad5 Vector, HIV-1 Inserts and Wild Type Ad5

Cellular DNA/RNA/cDNA

  • 1st round limiting dilution PCR to amplify:
  • Ad5 vaccine vector (Junction)
  • HIV-1 insert genes (gag/pol/nef)
          • Wild type Ad5 (E1 region)

hCMV

Promoter

HIV-1gag/pol/nef

Ad5

ITRR

ITRL

//

ΔE1

Junction

2nd round PCR utilizing Real Time PCR system

Screening for positives

Positive PCRs

Sequence the amplified Ad5 vaccine construct and HIV-1 inserts

Calculate the copy numbers

one copy detection of mrkad5gag pol nef
One Copy Detection of MrkAd5gag/pol/nef

1,000 E+1

HIV-1 gag/Vaccine Ad5

HIV-1 pol/Vaccine Ad5

pol

Junction

1,000

Junction

gag

Rn

1,000 E+1

HIV-1 nef/Vaccine Ad5

WT Ad5/Vaccine Ad5

nef

Junction

1,000

Junction

WT Ad5

0

30

40

0

30

40

10

10

20

20

Cycles

slide9

Detection and Sensitivity with New PCR Assay for Individual Plasmid

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).

slide11

Summary and Conclusions

  • This assay has the sensitivity to consistently detect one copy of Ad5 vaccine construct, HIV-1 gag/pol/nef and wild type Ad5 (determined by plasmid controls), and can potentially be used for large scale screening of vaccine trail study participants.
  • 25 samples (8 subjects) ranging from baseline to 56 weeks post 1st vaccination were screened with our new assay. There were no detectable Ad5 vaccine construct, HIV insert genes (gag/pol/nef) or wild type Ad5 from the 2-8 million PBMC tested.
slide12

Future Directions

    • The Ad5 vaccine vector and HIV-1 insert genes do not persist in PBMC of humans > 15 weeks post 1st vaccination. To determine how long they persist post vaccination (<15 weeks) requires further testing.
      • Testing samples drawn close to the time of vaccination (e.g., 1-7 days post vaccination)
      • Testing samples drawn between 1-15 weeks post vaccination.
  • Examination of other compartments (e.g., local tissue, lymph node, mucosal biopsy tissues) for persistence of vector.
acknowledgements
University of Washington

Tuofu Zhu Lab

Haiying Zhu

Dominic M. Forte

Luis Acevedo

Tom Andrus

Molecular Diagnostic lab

Meei-Li Huang

Larry Corey

Acknowledgements

HVTN Laboratory Program

Lynne A. Becker

Nicole Frahm

Ann Duerr

James Kublin

Steve Self

John Hural

Julie McElrath

Larry Corey

Volunteers

Merck

Andrew J. Bett

Danilo R. Casimiro