This document summarizes Dr. Catherine N. Kibirige's presentation on developing ultra-sensitive PCR protocols for HIV vaccine research. It discusses (1) the challenges of HIV's genetic diversity and finding conserved sequence regions for assay design, (2) how digital droplet PCR (ddPCR) was useful for quantifying cell line standards but qPCR was better for analyzing archival patient samples due to issues with ddPCR, and (3) two successful studies using the ultra-sensitive qPCR assay - a pilot study found HIV RNA more frequently in virologically suppressed patients with high inflammation, and characterization of the IAVI Viral Inhibition Assay showed HIV RNA detection within 2 days of differences in CD8 inhibition

1. Global Engage 5th qPCR and ddPCR Congress
Developing Ultra-Sensitive PCR Protocols
for HIV Vaccine Research
Dr. Catherine N. Kibirige
Hammersmith, London
5 December, 2017

2. Contents
➢ HIV Virology and Life Cycle
➢ HIV Viral Diversity
o – The challenge of finding homologous sequence regions
➢ Ultra-Sensitive (Ultra-S) Assay Design (qPCR versus ddPCR)
o The challenge of archival patient samples
➢ Success Stories:-
o Pilot Patient Study– Multi-Center AIDS Cohort Study
o Characterization of the IAVI Viral Inhibition Assay

3. Human Immunodeficiency Virus
Acquired Immunodeficiency syndrome – AIDS
➢ AIDS first described in 1981
➢ HIV-1 isolated in 1984
➢ Genus: Retroviridae
➢ Subfamily: Lentivirus
➢ Enveloped RNA Genome

4. The HIV Life Cycle
HIV RNA species
predominate
over
DNA

5. HIV Genome Diversity
Nucleotide sequence alignment of the
LTR U3 and U5 regions of 43 HIV-1
strains.
➢ HIV-1 provirus with the LTRs and the three
genes gag, pol, and env
o Dashes = identity.
o Periods = gaps in the nucleotide
sequence.
o Shaded region = location of the
conserved AT sequence
➢ Sequences obtained from http://hiv-
web.lanl.gov/
Nouri Neamati et al. Mol Pharmacol 1998;54:280-290

6. qPCR versus ddPCR
➢ ddPCR worked for the quantification of Cell Lines
and Standards but not for RNA or patient samples
cDNA synthesis of HIV LTR region problematic
1/10 cDNA dilution required
When sample copy numbers were very low - distorted by
noise
Nested PCR format created a lot of “rain”
➢ ddPCR highlighted grave discrepancies in HIV-1
copy number within cell line standards.
(8E5, J1.1 and U1 HIV-1 = 0.1 to 2.6 copies of HIV per cell )
➢ ddPCR was thus used to quantified standards
➢ qPCR was used to analyse archival patient
samples

7. Ultra-Sensitive HIV RTqPCR Assay
Nested PCR Format
➢ 1. HIV-specific cDNA + Pre-amplification
Tagged Conserved Forward Primer
Conserved Reverse Primer
➢ 2.Taqman PCR Cycling
Forward Primer against Tag
Internal Reverse Primer
Doubly-Quenched Probe
Kibirige et al., Journal of Virological Methods

8. Ultra-Sensitive HIV RTqPCR Assay
Limit of Detection ~3
~95% Confidence
Standard Curve
ThresholdCycle(CT)
Quantity (Copies)
Assay Performance
➢ Limit of Detection and Quantification
3 Input Copies of HIV RNA
95% Confidence
(Standard = 10 input copies)
➢ Linear Range ~< 10,000
Pilot study experiments run in triplicate
with separate reference gene analysis
Good results with cellular lysates of
<1,500 cells
Kibirige et al., Journal of Virological Methods

9. DEMONSTRATING CLINICAL UTILITY
Pilot Patient Study
Clinical Characteristics
➢ 18 virologically suppressed (<20 copies HIV-1 RNA/ml plasma)
HIV-positive men in the Multicenter AIDS Cohort Study (MACS)
➢ high or low inflammation (serum IL-6 and C-reactive protein
levels in the highest or lowest quartiles, respectively).
Cell Isolation
➢ CD4+CD27+CD45RA+ naïve, CD4+CD27+CD45RA- central
memory, and CD4+CD27-CD45RA+/- peripheral memory/effector

10. RESULTS
➢ HIV-1 RNA was detected significantly
more frequently in 21/27 (78%) of
samples from the high inflammation
group and 7/27 (26%) samples from the
low inflammation group (p=.0003).
o Central Memory Subset (p=.05)
➢ More CD4+CD45RA-CD27-CD28+ cells
in the high versus low inflammation
groups (74% vs 50%, p=.08)
Kibirige et al., Journal of Virological Methods
PILOT PATIENT STUDY

11. Days
RLU
CD4+CD8
CD4
IAVI VIRAL INHIBITION ASSAY (VIA)
For the Assessment of Functional Antiviral T-cell Responses

12. • The VIA assay measures HIV p24 antigen in culture supernatants at day 13
• An alternative assay measures Luciferase production at day 8
• The VIA is optimized for robust screening of vaccines
• Measuring HIV nucleic acids – particularly RNA – should allow for a more
sensitive assay read-out and more precise assessment of virus potency at
earlier time points
IAVI VIRAL INHIBITION ASSAY (VIA)
For the Assessment of Functional Antiviral T-cell Responses

13. REFERENCE GENE VALIDATION
+/- IL-2
• T cells are cultured in the presence
of IL-2 in the Standard VIA Assay
• IL-2 can affect the expression of
reference genes
• It is important to validate the best
reference genes for the assay

14. CHARACTERIZING RNA KINETICS IN THE VIRAL INHIBITION ASSAY
• HIV RNA compared well with p24 release as a VIA assay readout
• Differences in CD8 Inhibition can be detected within 2 days with HIV RNA

15. HIV VIRULENCE CORRELATES WITH RNA KINETICS
P=0.0386
*
Virulence Score
HIVRNA
(cps/ng)
With IL-2Without IL-2
Expanded CD4 T Cells 48h Post-Infection
• Measuring HIV RNA
allows for the
detection of
differences in viral
virulence within 48
hours
• Future experiments
will investigate
archival patient
samples and primary
T cell cultures

16. Multi-Center AIDS Cohort Study
Joseph Margolick
Hao Z. Zhang
Tricia A. Nilles
Bui Hanvyi
Fred Menendez
US Military HIV Research Program
Sheila Peel
Mark Manak
Linda Jagodzinski
Holly Hack
Ying Liu
International AIDS Vaccine Initiative
Human Immunology Laboratory
Jill Gilmour
Ed McGowan
Peter Hayes
Adriano Boasso
Weiwei Ma
Natalia Fernandez
Jama Dalel
Lucas Black
Ambreen Ashraf
Adam Coleman
ACKNOWLEDGEMENTS
FUNDING: US National Institutes of Health, US Military HIV Research Program, USAID