HIV Eradication - possible/feasible or not? Molecular Virology - - PowerPoint PPT Presentation

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 Vicenti1, Filippo Dragoni1, Martina Monti2,4, Alessia Giannini1, Annalisa Ciabattini1, Barbara Rossetti3, Andrea De Luca1,3, Donata Medaglini1 , Emanuele Montomoli2,4, and Maurizio Zazzi1

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)

• 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

• 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? Inten ensi sification and s nd simplification s stud udies. es.

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

• bserved 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

MVC: a : a drug w with th double u use? Backgr ground i in v vitro a and ex-vivo vo

• To define MVC activity as a latency reversing agent

using three cell line models in vitro Aim of f th the S Stu tudy

Methods (I)

Activation of HIV-1 LTR was determined by comparing the luciferase readout of treated vs untreated cells Cells treated with serial doses of MVC and with known LRAs (positive control)  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).

Untreated LTR

Luciferase

LTR

β-galactosidase

Treated LTR

Luciferase

LTR

β-galactosidase

INDUCTION for 24 h

LUMINESCENCE MEASUREMENT

TZM-bl Immortalized adherent cell line derived from Hela

Methods(II)

 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).

Induction of ACH-2 and U1/HIV-1 cell lines

STEP 1

INDUCTION for 24 hrs

SUPERNATANT & & CE CELL P PELLET COLLEC ECTION ON Latently HIV-1 infected lymphoblastoid T cell lines ACH2 and U1/HIV-1

Cells treated with serial doses of MVC and with known LRAs (positive control)

Methods (III)

 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/106 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).

STEP 2

Sample Lysis HIV RNA binding Wash RNA elution (Ready for RT)

Shan et al.,2013

Reverse Transcription HIV-1 RNA Extraction

Real Time PCR

• 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

Methods (IV)

RESULTS(I): Expression of CCR5

ACH-2 U1 TZM-bl CCR5

Normalized events Unlabelled cells CCR5 stained cells

% % %

RESULTS(II): TZM-bl cell line model

Luciferase Expression MVC (from 80 to 0.31 µM): 0.89±0.06 FA PHA: 1.00±0.06 FA ION+PMA: 4.31±0.14 FA

• 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)

D ru g C o n c e n tra tio n F o ld ac tivatio n

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 IO N + P M A 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 IO N + P M A P H A C C 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0

L U C IF E R A S E E X P R E S S IO N

T Z M -b l c e ll lin e

F O L D O F A C T IV A T IO N N F -k B

NF-kB Expression MVC (from 80 to 0.31 µM): 1.17 ±0.23 FA Minimal activation at MVC 5 µM: 1.63±0.40 FA PHA: 1.03±0.01 FA ION+PMA: 3.87±0.76 FA

RESULTS (III): ACH-2 cell line model

Extracellular HIV-1 RNA Activation at MVC 80µM (3.92±1.39 FA) Minimal activation at MVC 20µM (1.74±1.03 FA) PHA (0.65±0.45 FA) Cell-associated HIV-1 RNA Minimal activation at MVC 80µM (1.73±0.68 FA) PHA (1.31±0.59 FA) NF-kB NF-kB expression was not upregulated at any MVC concentration tested (0.60±0.11 FA)

• 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 F o ld a c tiv a tio n

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 IO N + P M A P H A C C 1 2 3 4 5 6 7 8 9 1 0 5 0 1 0 0 1 5 0 2 0 0

N F -kb C e ll a sso cia te d H IV -1 R N A E xtra ce llu la r H IV -1 R N A

A C H -2 c e ll lin e

1.5

RESULTS (IV): U1/HIV-1 cell line model

Extracellular HIV-1 RNA Activation at 80µM (3.11±0.92 FA) Minimal activation at 5µM (1.90±0.46) PHA (0.83±0.19 FA) Cell-associated HIV-1 RNA MVC at 20µM (1.40 ±0.39 FA) induces cell associated HIV-1 RNA similarly to PHA (1.30 ±0.37 FA) but with values below 1.5 FA NF-kB NF-kB expression was not upregulated at any MVC concentration tested

• Red line indicates the mock level
• A minimal activation is considered when FA≥1.5

Drug Concentration Fold activation

MVC 80 MVC 20 MVC 5 MVC 1,25 MVC 0,31 ION+PMA PHA CC 1 2 3 4 5 100 1100 2100 3100

NF-kb Cell associated HIV-1 RNA Extracellular HIV-1 RNA

U1/HIV-1 cell line

1.5

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