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

1
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

2
HVTN 071 Trial

• HVTN 071 was a trial administering MRKAd5
  gag/pol/nef vaccine, designed to test the 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?

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HVTN 071 Vaccination Schedule
Weeks 52-56
PBMC or Leukapheresis (n8)
PBMC or Leukapheresis (n8)
PBMC or Leukapheresis (n8)
PBMC or Leukapheresis (n1)
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Objectives

1. 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.
2. 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.

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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 323208-209, 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
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One Copy Detection of Extraordinarily Low Levels
of HIV-1 Infection
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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
//
?E1
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
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One Copy Detection of MrkAd5gag/pol/nef
1,000 E1
HIV-1 gag/Vaccine Ad5
HIV-1 pol/Vaccine Ad5
pol
Junction
1,000
Junction
gag
Rn
1,000 E1
HIV-1 nef/Vaccine Ad5
WT Ad5/Vaccine Ad5
nef
Junction
1,000
Junction
WT Ad5
0
10
30
40
0
10
30
40
20
20
Cycles
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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.,
59505-511, Biometrics 2003).
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PCR Detection Results
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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.

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Future Directions

• The Ad5 vaccine vector and HIV-1 insert genes do
  not persist in PBMC of humans gt 15 weeks post 1st
  vaccination. To determine how long they persist
  post vaccination (lt15 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.

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Acknowledgements
HVTN Laboratory Program Lynne A. Becker Nicole
Frahm Ann Duerr James Kublin Steve Self John
Hural Julie McElrath Larry Corey Volunteers

• University of Washington
• Tuofu Zhu Lab
• Haiying Zhu
• Dominic M. Forte
• Luis Acevedo
• Tom Andrus
• Molecular Diagnostic lab
• Meei-Li Huang
• Larry Corey

Merck Andrew J. Bett Danilo R. Casimiro