pilot study goals
DESCRIPTION
Cerebrospinal Fluid (CSF) Drug Resistant HIV Compartmentalization Detected during Primary HIV-1 Infection by Ultra Deep Sequencing. - PowerPoint PPT PresentationTRANSCRIPT
Cerebrospinal Fluid (CSF) Drug Resistant HIV Compartmentalization
Detected during Primary HIV-1 Infection by Ultra Deep Sequencing
Arjet Gega1, Michael Kozal1, Jennifer Chiarella1, Evelyn Lee2, Julia Peterson2, Elizabeth St. John3, Birgitte Simen3, Richard W. Price2 and Serena Spudich1
1Yale University School of Medicine, New Haven, CT, USA; 2University of California San
Francisco, San Francisco, CA, USA; 3454 Life Sciences, Branford, CT, USA.
Pilot Study Goals
• Demonstrate feasibility of ultra deep sequencing (UDS) of HIV species derived from cerebrospinal fluid (CSF)
• Use UDS to investigate early CNS compartmentalization of HIV
• Examine for low abundance transmitted drug resistant variants in early CNS-derived HIV
Background• Prior methods (heteroduplex tracking assay, single
genome amplification, Sanger sequencing) have shown compartmentalization of CSF-derived HIV RNA species
– CNS compartmentalization of env strongly associated with HIV associated dementia in advanced disease
– CSF compartmentalization detected in minority of subjects during primary infection
• Recent studies suggest relevance of drug resistant HIV species in CSF for CNS disease in setting of cART
• Presence of low abundance transmitted drug resistant variants in plasma predicts systemic virologic failure in setting of cART
Ritola J Virology 2005; Schnell J Virology 2010, Canestri CID 2010, Bogoch J Infection 2011, Simen JID 2009
HIV RNA in Plasma and CSF in Primary Infection
HIV RNA Levels
0 100 200 3001
2
3
4
5
6
7
8
Days Post HIV Transmission
plasma
log
10 c
opie
s/m
l
Spudich et al., JID, In press.
HIV RNA in Plasma and CSF in Primary Infection
HIV RNA Levels
0 100 200 3001
2
3
4
5
6
7
8
Days Post HIV Transmission
plasmaCSF
log
10 c
opie
s/m
l
Spudich et al., JID, In press.
Characteristics of Study Subjects
ID Sex Age Days post HIV
Transmission
CD4(cells/ul)
CD8(cells/ul)
Plasma HIV RNA
(log10 copies/ml)
CSF HIV RNA
(log 10 copies/ml)
CSF WBC
(cells/ul)
CSF/Serum
Albumin
9055 M 32 104 619 924 5.38 3.51 12 6.42
9039 M 33 36 539 1591 5.57 4.30 53 -
9044 M 25 126 533 575 4.82 3.49 20 4.30
9024 M 33 217 974 1840 4.75 4.29 12 5.74
9058 M 29 109 237 872 5.18 3.67 4 2.44
All subjects MSM from San Francisco, USA, enrolled between 2008 and 2010
Viral Quasispecies
Drug Resistant Viral Variants Detected by Ultra Deep Sequencing
0102030405060708090
100
% o
f HIV
var
iant
s
Population based Sanger Sequencing
Ultra-deepSequencing
NNRTI resistant
WT viral variants
Within-host virus population consists of different viral variants that are evolutionarily related.
Kozal MJ. Next Gen Dx Summit. Washington DC 2010 & Paredes R. Asian Pasic Next Gen Conf. Hong Kong 2010
New technologies provide the ability to sequence unique individual viral genomes
Read Flowgram
Ultra Deep Sequencing
Mix amplicons & capture beads
Isolate DNA containing beads
PCR in “water-in-oil”
emulsion
Add PCR Reagents& emulsion oil
Amplicon pool
A
B
Micro-reactors
Load Enzyme Beads
Load beads onto PicoTiter™Plate
DNA Capture
Bead T
ATP
Light + oxyluciferin
Sulfurylase
Luciferase
APS
luciferin
PPi
Load PTP on Sequencer
Pyro-sequence
Gega & Kozal. Future Virology 2011
Subject 9055: 104 Days Post Transmission
VL PI RTReads %
Mut Var
PI ML Codon Reads
Reads % Mut Var
RT ML CodonReads
Plasma5.38log
8206 Wild Type 2876 Wild Type
CSF3.51log
6995 Wild Type 2369 Wild Type
32 yo MSM, CD4 619 CSF WBC 12, CSF/Serum Albumin 6.42
VL: HIV RNA level% Mut Var: % mutant variants detected by UDSML: estimated mutational load = mutant variant frequency x HIV RNA level (VL)
Subject 9039: 36 Days Post Transmission
VL PI RTReads % Mut
VarPI ML Codon
ReadsReads % Mut
VarRT ML
CodonReads
Plasma4.75log
6368 D30N(0.69) V82A(0.3)N83D(0.3) I85V(0.27)
D30N(388) V82A(169) N83D(169)I85V(152)
D30N (6,378)V82A
(6,589)N83D
(8,599)I85V
(8,599)
2524 Wild Type
CSF4.29log
7471 Wild Type 2575 Wild Type
33 yo MSM, CD4 539, CSF WBC 53
Subject 9044: 126 Days Post Transmission
VL PI RTReads % Mut
VarPI ML Codon
ReadsReads % Mut
VarRT ML
CodonReads
Plasma4.82log
7237 M46I(0.46)I47V(1.16)
M46I(304) I47V(766)
M46I (6,130) I47V
(6,130)
2876 Wild Type
CSF3.49log
8645 V82A(0.39)
V82A(12)
V82A(10,644)
4394 Wild Type
25 yo MSM, CD4 533, CSF WBC 20, CSF/Serum Albumin 4.3
Subject 9024: 217 Days Post Transmission
VL PI RTReads % Mut
VarPI ML Codon
ReadsReads % Mut
VarRT ML Codon
ReadsPlasma
5.57log
7352 Wild Type 1381 M184V*(0.33)T215D*(99.41)T227L (0.42)
M184V(1,226)T215D(369K)T227L (1,560)
M184V (1,196)T215D (1,196)T227L (1,196)
CSF4.3log
6608 V82A(0.52)
N83D(0.27)
V82A(104)
N83D(54)
V82A (7,917)
N83D (7,917)
2396 F77L(0.29)T215D*(99.43)T227L*(0.26)
F77L(58)T215D(19,838)T227L(52)
F77L (3,095) T215D (2,277) T227L (2,277)
33 yo MSM, CD4 974, CSF WBC 12, CSF/Serum Albumin 5.74
*linkage present
Subject 9058: 109 Days Post Transmission
VL PI RTReads % Mut
VarPI ML Codon
ReadsReads
% Mut Var
RT ML CodonReads
Plasma5.18log
6567 Wild Type 2642 D67G(0.44) T69N(1.31) G190E(0.34) T227L(0.27)
D67G(666) T69N
(1,983) G190E(515)
T227L(409)
D67G (2,972) T69N
(2,972) G190E (2,619) T227L (2,619)
CSF3.37log
5582 D30N*(9.77) M46I*(8.1) G73S( 5.16) V82A*(0.27)
D30N(451) M46I(374) G73S( 238) V82A(12)
D30N (4,604) M46I
(4,604) G73S (6,589) V82A
( 6,589)
2628 L210W(0.59)G190E(1.56)
L210W(27)
G190E(72)
L210W* (2,891)G190E* (2,891)
29 yo MSM, CD4 237, CSF WBC 4, CSF/Serum Albumin 2.44
*linkage present
Plasma HIV RNA
0 250 500 750 10000
1
2
3
4
5
6
9039
90249044
9055
9058lo
g 10
cop
ies/
ml
CSF HIV RNA
0 250 500 750 10000
1
2
3
4
5
6
Days Post HIV Transmission
log
10 c
opie
s/m
l
Longitudinal Plasma and CSF HIV RNA Levels
TDF/FTC/RAL
DRV/RTV/RAL
TDF/FTC/DRV/RTV
Limitations• Pilot, ‘proof of concept’ study with 5 subjects only• Subjects with high CSF HIV RNA picked from cohort• 0.2% chosen as lowest detection level as >3x greater than
PCR error rate:– Error occurring in early rounds of amplification may still affect
results
• Mutational load values only an estimate:– Calculated by multiplying the results from separate molecular
assays using different primer sets
• As sample HIV RNA level decreases, the ability to obtain a fully representative sample of HIV RNA templates declines: – Levels of mutations may represent the proportion of sequenced
PCR amplicons containing the mutation vs. the actual proportion in the sample
Conclusions
• UDS of HIV from CSF samples was successful in 5 subjects with CSF HIV RNA > 3000 copies/ml.
• Marked differences can exist between quasispecies in blood and CSF at a median < 4 months after HIV transmission.
• Four patients had substantial differences in resistance mutations in plasma and CSF HIV variants which could potentially impact clinical response to cART.
• Purely descriptive study; larger and longitudinal studies are needed to determine clinical significance.
AcknowledgementsUCSF/San Francisco:• Study Volunteers• Richard W. Price • Evelyn Lee• Julia Peterson• Frederick Hecht• Christopher Pilcher• UCSF Options Study Staff• Magnet Staff/Volunteers• Teri Liegler• SFGH/GIVI Virology Lab
Yale: • Arjet Gega• Michael Kozal• Jennifer Chiarella
454 Life Sciences:• Birgitte Simen• Elizabeth St. John• Elizabeth Moreno
NIH/NIMH/NIAIDSF AIDS Research InstituteUCSF REAC/Academic Senate