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Dr Isabella Abbate Laboratory of Virology National Institute for Infectious Diseases

Quasispecies analysis by ultra-deep pyrosequencing of HIV-1 deriving from lymphomonocyte sub-populations in chronically infected patients at the moment of HAART interruption. Dr Isabella Abbate Laboratory of Virology National Institute for Infectious Diseases “L. Spallanzani”, Rome Italy.

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Dr Isabella Abbate Laboratory of Virology National Institute for Infectious Diseases

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  1. Quasispecies analysis by ultra-deep pyrosequencing of HIV-1 deriving from lymphomonocyte sub-populations in chronically infected patients at the moment of HAART interruption Dr Isabella Abbate Laboratory of Virology National Institute for Infectious Diseases “L. Spallanzani”, Rome Italy

  2. Background • V3 loop region of gp120 is the most variable region of HIV-1 (involved in coreceptor binding) • Compartimentalized viral replication in different body sites and target cells may lead to divergent virus evolution • So far, studies on cellular compartimentalization of HIV-1 have been performed by analyzing proviral DNA quasispecies • Cell membrane proteins on HIV-1 are markers of virus cell origin • Antibodies against these molecules can be used to sort virus originating ex vivo from a particular cell reservoirs

  3. Aim To analyze the quasispecies of proviral and viral HIV-1 originating from monocytes and T lymphocytes by ultra-deep pyrosequencing Experimental design Patients with prolonged successful HAART were selected, to highlight possible divergent evolution

  4. Patients The enrolled subjects were five chronically HIV-1 infected patients who underwent therapy interruption after >5 years of successful HAART.

  5. Methods Quasispeciesanalysisperformedbyultra-deeppyrosequencing on: Provirus:Cellassociated HIV-1 from CD36 (monocytes) and CD26 (T lymphocytes) at HAART interruption Virus: Plasma HIV-1 originatingex vivofrommonocytes and T lymphocytescapturedbyantibodies anti CD36 and CD26 (1 monthafter HAART interruption) CD36 gp120 others CMPs MAb to CD36 Goat anti-mouse • The env region examined: 66 aa (-16 before and +15 after V3 loop) • Sequencesobtainedbypyro-sequencingwereusedto: • Calculateheterogeneityparameters • Predictcoreceptorusageby PSSM analysis • Constructphylogenetictrees

  6. Traditionalapproachesforquasispeciesanalysis: 1. Cloningof PCR amplicons and sequencing via Sanger’s method 2. Limitingdilution PCR and sequencing via Sanger’s method

  7. Ultra-deep pyrosequencing by genome sequencer (GS-FLX Roche technology) Allows to simultaneously analyze thousands of clonally amplified amplicons

  8. Comparison between GS-FLX technology and the traditional cloning and sequencing approach for phylogenetic tree construction

  9. Results 1. V3 loop aminoacid diversity and complexity of HIV-1 from T lymphocytes and monocytes as obtained by ultra-deep pyrosequencing * p=0.027 CD36 vs CD26 ** p=0.160 CD36 vs CD26 41,480

  10. Results 2.PSSM scores for prediction of coreceptor usage of HIV-1 deriving from T lymphocytes and monocytes Score ofreference R5 strain BaL -12.96 “ “ “ X4 “ HXB2 +3.47

  11. Results 3. Individual phylogenetic trees Unique variants Total sequences 98 86 89 99 Provirus CD36 59 CD26 and 231 CD36 unique variants on total 8723 sequences Provirus CD26

  12. Pt.1 Score: -10.25 Frequency: 100% 98 86 -9,78 Score: +3.73 Frequency: 1.4% 89 Score: -10.33 Frequency: 0.12% 99 Score: -10.90 Frequency: 0.23% Provirus CD36 Provirus CD26 0.001

  13. Pt.3

  14. Pt.4

  15. Pt.5 99 Score: -9.02 Frequency: 6.7% Provirus CD36 Provirus CD26 Virus CD36 0.005 Virus CD26

  16. Conclusions: • Higher V3 heterogeneity in monocytes as compared to lymphocytes • Proviral and circulating virus, from both cell types, are predominantly R5 • Very divergent provirus variants, including X4, may be present in monocytes • Possible therapeutic implications? • Both monocytes and T lymphocyte may contribute to virus rebounding after TI, but other virus sources may be involved Immunocapture of circulating virions and ultra-deep sequence analysis: powerful tool to investigate viral dynamics and to explore the contribution of different reservoirs to viremia

  17. Thanksto: Dr. Gabriella Rozera Laboratory of Virology, INMI L.Spallanzani Dr. Giuseppe Ippolito Dr. M.R. Capobianchi INMI L.Spallanzani LaboratoryofVirology,INMI L.Spallanzani Dr. Alessandro Bruselles Dr. Giovanni Chillemi LaboratoryofVirology,INMI L.Spallanzani Caspur Roma Dr. Pasquale Narciso Dr. Gianpiero D’Offizi Dr. Chrysoula Vlassi IV Clinical Division, INMI L.Spallanzani

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