Thoughts on a new subject: HIV Eradication - possible/feasible or not? Molecular Virology Group University of Basel Department Biomedicine - Petersplatz
This Session: • Maraviroc as a Latency Reversing Agent in cell line models (Ilaria Vicenti) • Eliminate HIV from the most crucial compartment! (Fabian Otte) • Which body compartment is represented in the circulation? • Where do long-lived HIV reservoirs really reside? • Why do 50% of the immune system stay under fire – even during therapy? • Extinguish HIV-infected cells that ’misbehave’! (Nina Marty) • Can long-term HIV infection induce cellular transformation ? • How do X4 and R5 tropism molecularly evolve during therapy? • Eradicate HIV – optimize treatment of populations most in need! (Jenny Brown) • How can we win the global 90-90-90 race if we lose the most vulnerable ? • On molecular drivers of HIV during “non-compliance” and interrupted therapy.
Maraviroc as a Latency Reversing Agent in cell line models Ilaria Vicenti 1 , Filippo Dragoni 1 , Martina Monti 2,4 , Alessia Giannini 1 , Annalisa Ciabattini 1 , Barbara Rossetti 3 , Andrea De Luca 1,3 , Donata Medaglini 1 , Emanuele Montomoli 2,4 , and Maurizio Zazzi 1 1 Department of Medical Biotechnologies, University of Siena, Siena, Italy 2 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 3 Infectious Diseases Unit, Azienda Ospedaliera Universitaria Senese, Siena, Italy 4 VisMederi srl, Siena, Italy Arevir Meeting, Cologne 2019
CCR5 Antagonist: MARAVIROC (MVC) The image part with relationship ID rId4 was not found in the file. • MVC is the only host-targeting anti-HIV agent, licensed for use in second-line regimen • MVC binds to the external part of the CCR5 transmembrane pocket inhibiting virus internalization • MVC has been reported to be associated with additional immunomodulatory effects in some studies • Decreased immune activation and inflammation markers (Funderburg et al., 2010) • Increased CD4 cell counts even in the context of virological failure (Asmuth et al., 2010) • Larger recovery of CD4 cells with respect to darunavir/ritonavir, both coupled with raltegravir and etravirine (Cossarini et al., 2012)
MVC: a drug with double use? Kim, Cell Host & Microbe 2018 23, 14-26
MVC: a : a drug w with th double u use? Inten ensi sification and s nd simplification s stud udies. es. • Beneficial effects of MVC on immunological activation in intensification studies (Gutierrez 2011, Wilkin 2012, Cillo 2015) • No changes in HIV-1 reservoir were observed when MVC was used in combination with other drugs and/or immunomodulatory factors (Ananworanich 2015, Katlama 2016, Lafeuillade 2014, Ostrowski 2015) • Reduction in HIV-1 reservoir following intensification of treatment with MVC alone: • In patients with recent HIV-1 infection (Puertas et al., 2014) • In patients with suppressed viremia (Gutierrez et al., 2011) • LRA like activity could explain HIV relapses occurred at low level and/or transiently in MVC simplification studies (Pett 2016, Rossetti 2017)
MVC: a : a drug w with th double u use? Backgr ground i in v vitro a and ex-vivo vo Lopez-Huertas, Sci Rep 2017: MVC activates the transcription of luciferase gene in vitro, under LTR control following transfection of the LTR-luc construct into 293 cells MVC activates HIV production (HIV Ca-p24) in a model of HIV latency in CD4+ cells in vitro Symons, IAC 2018 and CROI 2015: MVC in vivo and ex-vivo leads to an increase in cell associated HIV-1 RNA in CD4+ cells; dose-dependent increase in HIV production (HIV Ca-p24) was observed when MVC was added to PBMCs (2.2 fold) Madrid-Elena, J Virol 2018: MVC increased unspliced HIV RNA levels in vivo in resting CD4+ cells in association with enhanced expression of NF- κB dependent genes
Aim of f th the S Stu tudy To define MVC activity as a latency reversing agent using three cell line models in vitro
Methods (I) Cells treated with serial doses of MVC and with known LRAs (positive control) INDUCTION for 24 h Untreated LTR Luciferase LTR β -galactosidase LUMINESCENCE MEASUREMENT Treated Activation of HIV-1 LTR was LTR determined by comparing the Luciferase TZM-bl luciferase readout of treated LTR Immortalized adherent cell β -galactosidase vs untreated cells line derived from Hela MVC concentrations used were 80µM, 20µM, 5µM, 1.25µM, 0.31µM. Positive LRA controls were Ionomycin (1µg/ml) +PMA (50ng/ml) and PHA (10µg/ml). Results were expressed as Fold of activation (FA), which are the ratio between induced and not induced cell line, treated with DMSO (mock).
Methods(II) STEP 1 Induction of ACH-2 and U1/HIV-1 cell lines Cells treated with serial doses of MVC and with known LRAs (positive control) INDUCTION SUPERNATANT & & CE CELL P PELLET for 24 hrs COLLEC ECTION ON Latently HIV-1 infected lymphoblastoid T cell lines ACH2 and U1/HIV-1 MVC concentration used were 80µM, 20µM, 5µM, 1.25µM, 0.31µM. Positive LRA controls were Ionomycin (1µg/ml) +PMA (50ng/ml) and PHA (10µg/ml).
Methods (III) STEP 2 Real Time PCR HIV-1 RNA Extraction Reverse Transcription Sample Lysis HIV RNA binding Wash RNA elution (Ready for RT) Shan et al.,2013 HIV-1 induction was assessed by measuring HIV-1 RNA in the supernatant (HIV-1 RNA cp/ml) & in cell pellet (HIV-1 RNA cp/10 6 cell) by quantitative real time PCR. HIV-1 RNA was quantitated with downstream primers carrying oligo(dT) (Shan et al , 2013) in order to gain specific amplification of cDNA generated by reverse transcription of viral mRNA. Results were expressed as Fold of activation (FA), which are the ratio between induced and not induced cell line, treated with DMSO (mock).
Methods (IV) • NF- kβ induction was evaluated in all cellular nuclear extracts by the NF-kB (p65) Transcription Factor Assay Kit (ELISA) • Expression of CCR5 in the cell lines tested was assessed by flow cytometric analysis
RESULTS(I): Expression of CCR5 Unlabelled cells ACH-2 U1 TZM-bl CCR5 stained cells Normalized events % % % CCR5
RESULTS(II): TZM-bl cell line model T Z M -b l c e ll lin e L U C IF E R A S E E X P R E S S IO N F O L D O F A C T IV A T IO N N F -k B Luciferase Expression 5 .0 MVC (from 80 to 0.31 µM): 0.89 ±0.06 FA 4 .5 PHA: 1.00 ±0.06 FA 4 .0 ION+PMA: 4.31 ±0.14 FA 3 .5 F o ld ac tivatio n 3 .0 2 .5 NF-kB Expression 2 .0 MVC (from 80 to 0.31 µM): 1.17 ±0.23 FA 1 .5 Minimal activation at MVC 5 µM: 1 .0 1.63±0.40 FA 0 .5 PHA: 1.03 ±0.01 FA 0 .0 ION+PMA: 3.87 ±0.76 FA M V C 8 0 M V C 2 0 M V C 5 M V C 1 ,2 5 M V C 0 ,3 1 P H A C C M V C 8 0 M V C 2 0 M V C 5 M V C 1 ,2 5 M V C 0 ,3 1 P H A C C IO N + P M A IO N + P M A D ru g C o n c e n tra tio n • Red line indicates the mock level • A minimal activation is considered when FA≥ 1.5 • No effects of MVC on LTR and NF-kb expression at all the concentrations tested (80, 20, 5, 1.25 and 0.31 µM)
RESULTS (III): ACH-2 cell line model N F -kb Extracellular HIV-1 RNA C e ll a sso cia te d H IV -1 R N A A C H -2 c e ll lin e Activation at MVC 80µM ( 3.92 ±1.39 FA) E xtra ce llu la r H IV -1 R N A Minimal activation at MVC 20µM ( 1.74 ±1.03 FA) 2 0 0 1 5 0 PHA ( 0.65 ±0.45 FA) 1 0 0 5 0 1 0 Cell-associated HIV-1 RNA 9 Minimal activation at MVC 80µM ( 1.73 ±0.68 FA) F o ld a c tiv a tio n 8 PHA ( 1.31 ±0.59 FA) 7 6 5 NF-kB 4 NF-kB expression was not upregulated at any MVC 3 concentration tested ( 0.60 ±0.11 FA) 2 1.5 1 0 M V C 8 0 M V C 2 0 M V C 5 M V C 1 ,2 5 M V C 0 ,3 1 P H A C C IO N + P M A Red line indicates the mock level • • A minimal activation is considered when FA≥ 1.5 D ru g C o n c e n tra tio n
RESULTS (IV): U1/HIV-1 cell line model NF-kb Extracellular HIV-1 RNA U1/HIV-1 cell line Cell associated HIV-1 RNA Activation at 80µM ( 3.11 ±0.92 FA) Extracellular HIV-1 RNA Minimal activation at 5µM ( 1.90 ±0.46) 3100 PHA ( 0.83 ±0.19 FA) 2100 1100 Cell-associated HIV-1 RNA MVC at 20µM ( 1.40 ±0.39 FA) induces cell associated Fold activation 100 5 HIV-1 RNA similarly to PHA ( 1.30 ±0.37 FA) but with values below 1.5 FA 4 3 NF-kB 2 NF-kB expression was not upregulated at any MVC 1.5 1 concentration tested 0 MVC 80 MVC 20 MVC 5 MVC 1,25 MVC 0,31 ION+PMA PHA CC • Red line indicates the mock level • A minimal activation is considered when FA≥ 1.5 Drug Concentration
Summary NA, not applicable NT, not tested • MVC effects were generally weak (mostly at highest 80µM dosing) but comparable with PHA induction • Dose-response curves were inconsistent
Conclusion Based on these and previously published data, MVC is a weak inducer of HIV-1 expression in some but not all in vitro models of HIV- 1 latency Multiple modes of measurement are necessary to provide a full picture of the mechanisms(s) underlying MVC induction Ex vivo studies based on clinical samples from patients with controlled HIV-1 infection are necessary to elucidate the potential of MVC, if any, as an agent with a double mechanism of action
Thank you
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