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

op

ies/

ml

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

op

ies/

ml

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

0

10

20

30

40

50

60

70

80

90

100

% o

f H

IV v

aria

nts

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 RT

Reads % 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 RT

Reads % Mut Var

PI ML CodonReads

Reads % Mut Var

RT 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 RT

Reads % Mut Var

PI ML CodonReads

Reads % Mut Var

RT 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 RT

Reads % Mut Var

PI ML CodonReads

Reads % Mut Var

RT ML CodonReads

Plasma5.57log

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 RT

Reads % Mut Var

PI ML CodonReads

Reads

% 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 1

0 co

pie

s/m

l

CSF HIV RNA

0 250 500 750 10000

1

2

3

4

5

6

Days Post HIV Transmission

log

10

cop

ies/

ml

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