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Pilot Study Goals

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

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Pilot Study Goals

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  1. 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.

  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 All subjects MSM from San Francisco, USA, enrolled between 2008 and 2010

  7. Drug Resistant Viral Variants Detected by Ultra Deep Sequencing New technologies provide the ability to sequence unique individual viral genomes Population based Sanger Sequencing Ultra-deep Sequencing Viral Quasispecies WT viral variants % of HIV variants • Within-host virus population consists of different viral variants that are evolutionarily related. NNRTI resistant Kozal MJ. Next Gen Dx Summit. Washington DC 2010 & Paredes R. Asian Pasic Next Gen Conf. Hong Kong 2010

  8. Ultra Deep Sequencing Micro-reactors A Add PCR Reagents & emulsion oil B Isolate DNA containing beads PCR in “water-in-oil” emulsion Amplicon pool Mix amplicons & capture beads Load beads onto PicoTiter™Plate Load Enzyme Beads Load PTP on Sequencer DNA Capture Bead Read Flowgram Pyro-sequence T PPi APS Sulfurylase Luciferase ATP luciferin Light + oxyluciferin Gega & Kozal. Future Virology 2011

  9. Subject 9055: 104 Days Post Transmission 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 33 yo MSM, CD4 539, CSF WBC 53

  11. Subject 9044: 126 Days Post Transmission 25 yo MSM, CD4 533, CSF WBC 20, CSF/Serum Albumin 4.3

  12. Subject 9024: 217 Days Post Transmission 33 yo MSM, CD4 974, CSF WBC 12, CSF/Serum Albumin 5.74 *linkage present

  13. Subject 9058: 109 Days Post Transmission 29 yo MSM, CD4 237, CSF WBC 4, CSF/Serum Albumin 2.44 *linkage present

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