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Cerebrospinal Fluid (CSF) Drug Resistant HIV Compartmentalization Detected during Primary HIV-1 Infection by Ultra Deep Sequencing Arjet Gega 1, Michael.

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Presentation on theme: "Cerebrospinal Fluid (CSF) Drug Resistant HIV Compartmentalization Detected during Primary HIV-1 Infection by Ultra Deep Sequencing Arjet Gega 1, Michael."— Presentation transcript:

1 Cerebrospinal Fluid (CSF) Drug Resistant HIV Compartmentalization Detected during Primary HIV-1 Infection by Ultra Deep Sequencing Arjet Gega 1, Michael Kozal 1, Jennifer Chiarella 1, Evelyn Lee 2, Julia Peterson 2, Elizabeth St. John 3, Birgitte Simen 3, Richard W. Price 2 and Serena Spudich 1 1 Yale University School of Medicine, New Haven, CT, USA; 2 University of California San Francisco, San Francisco, CA, USA; 3 454 Life Sciences, Branford, CT, USA.

2 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

3 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

4 HIV RNA in Plasma and CSF in Primary Infection Spudich et al., JID, In press.

5 HIV RNA in Plasma and CSF in Primary Infection Spudich et al., JID, In press.

6 Characteristics of Study Subjects IDSexAge 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 321046199245.383.51126.42 9039 M 333653915915.574.3053 - 9044 M 251265335754.823.49204.30 9024M3321797418404.754.29125.74 9058 M 291092378725.183.6742.44 All subjects MSM from San Francisco, USA, enrolled between 2008 and 2010

7 Viral Quasispecies Drug Resistant Viral Variants Detected by Ultra Deep Sequencing % of HIV variants Population based Sanger Sequencing Ultra-deep Sequencing NNRTIresistant 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

8 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

9 Subject 9055: 104 Days Post Transmission VLPIRT Reads% Mut Var PI MLCodon Reads Reads% Mut Var RT MLCodon Reads Plasma 5.38 log 8206Wild Type2876Wild Type CSF 3.51 log 6995Wild Type2369Wild Type 32 yo MSM, CD4 619 CSF WBC 12, CSF/Serum Albumin 6.42 VL: HIV RNA level % Mut Var: % mutant variants detected by UDS ML: estimated mutational load = mutant variant frequency x HIV RNA level (VL)

10 Subject 9039: 36 Days Post Transmission VLPIRT Reads% Mut Var PI MLCodon Reads % Mut Var RT ML Codon Reads Plasma 4.75 log 6368D30N (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) 2524Wild Type CSF 4.29 log 7471Wild Type2575Wild Type 33 yo MSM, CD4 539, CSF WBC 53

11 Subject 9044: 126 Days Post Transmission VLPIRT Reads% Mut Var PI MLCodon Reads % Mut Var RT ML Codon Reads Plasma 4.82 log 7237M46I (0.46) I47V (1.16) M46I (304) I47V (766) M46I (6,130) I47V (6,130) 2876Wild Type CSF 3.49 log 8645V82A (0.39) V82A (12) V82A (10,644) 4394Wild Type 25 yo MSM, CD4 533, CSF WBC 20, CSF/Serum Albumin 4.3

12 Subject 9024: 217 Days Post Transmission VLPIRT Reads% Mut Var PI MLCodon Reads % Mut Var RT MLCodon Reads Plasma 5.57 log 7352Wild Type1381M184V* (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) CSF 4.3 log 6608V82A (0.52) N83D (0.27) V82A (104) N83D (54) V82A (7,917) N83D (7,917) 2396F77L (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

13 Subject 9058: 109 Days Post Transmission VLPIRT Reads% Mut Var PI MLCodon Reads % Mut Var RT MLCodon Reads Plasma 5.18 log 6567Wild Type2642D67G (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) CSF 3.37 log 5582D30N* (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) 2628L210W (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

14 Longitudinal Plasma and CSF HIV RNA Levels TDF/FTC/RAL DRV/RTV/RAL TDF/FTC/DRV/RTV

15 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

16 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.

17 Acknowledgements UCSF/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/NIAID SF AIDS Research Institute UCSF REAC/Academic Senate


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