Candidate Microbicides: What we can learn from in vitro work Guido Vanham, MD PhD [email protected]...
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Transcript of Candidate Microbicides: What we can learn from in vitro work Guido Vanham, MD PhD [email protected]...
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Candidate Microbicides:
What we can learn from in vitro work
Guido Vanham, MD [email protected]
Institute of Tropical Medicine, Antwerp
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Vaginal HIV transmission
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TRANSCRIPTION +TRANSLATION
REVERSETRANSCRIPTASE
INHIBITORS
BINDINGINHIBITORS
FUSIONINHIBITORS
INTEGRASEINHIBITORS
ssRNA
dsDNA
HIV
TARGETCELL
CD-4CCR-5
ITM – Y. Van Herrewege
Direct DISRUPTION
HIV life CyclePotential targets for prevention
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Possible classes of candidate microbicides
- Buffers: Acidform, Buffergel: still in trial but only indirect antiviral action – Virus disrupters: Nonoxynol-9, Savvy (C31G) = obsolete
– Non-specific binding inhibitors: Cellulose sulphate; Carraguard, PRO-2000; Vivagel
some failed, some still in trial but even in vitro weak anti-HIV activity – Inhibitors of gp120:CD4 (e.g. BMS806, BMS793)– Inhibitors gp120:CCR5 (e.g. TAK-779, Maraviroc)– Inhibitors of gp120: DC SIGN (e.g. Mannan)
– Fusion inhibitors (e.g. T20, D-peptides)
– Reverse Transcriptase inhibitors (RTI): in trial Nucleotide RTI: PMPA (Tenofovir) + FTC (Truvada) NNRTI: TMC120 (Dapivirin), UC781
– Integrase inhibitors e.g. L 870 812 (Raltegravir analogue) ?
– Protease inhibitors e.g. Saquinavir ?
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In Vitro
Activity against pathogen
Cellular toxicity profile
Animal models
Safety: - Rabbit vaginal irritation
Efficacy to prevent infection: - NOD/SCID-PBL mice: HIV- Macaques: (SIV or SHIV)
Human (clinical)
Safety - In low-risk women (Phase I)- In representative population (Phase I/II)
Effectiveness (Phase III)
10 + years
Clinical Research Process
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In Vitro models to test HIV Microbicides
- Limited access (HT -)- Risk of damaging epithelium
Main advantages Main disadvantages
CD4/CCR5 (+) cell lines e.g. GHOST, U87, TZMbl
- Rapid screening- Single cycle virus (no L3 required)
Cells not representative for in vivo targets
Mitogen activated PBMC - Standard system- Relatively quick + easy
Only activated T cells
Co-cultures of dendritic cells and T cells (DC/T4)
More representative for primary targets in sexual mucosa
More complex and time consuming manipulation
DC/T4 + epithelial cells Additional relevance Additional complexity
Cervico-vaginal explant Most representative - Limited access- Limited viability- Epithelium not intact = corresponds to worst case in vivo scenario
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Data on candidate microbicides in in vitro models
1) Cell suspension models
A) Cell line (GHOST) + single cycle pseudovirus
B) DC/T4 co-culture: monocyte-derived dendritic cells + autologous T4 cells + primary replicative virus
2) Models of female genital tract mucosa
A) In vitro dual chamber model : DC/T4 + epithelial cells on top
B) Ex vivo cervico-vaginal explant = tissue from hysterectomy
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“Microbicide” Ba-L PV
+
30’
Ghost-CD4-CCR5
48h
Pre-incubation InfectionProduction of
luciferase
55
TAK779 42
T20
Binding gp120:CCR5
Fusion
Reverse transcription PMPA
UC781TMC120
L870812
82 2 7
9
Compound EC50 (nM) gmeanMechanism of inhibition
Binding gp120:CD4 BMS806 5
Integrase
Pseudovirus Test = Screening
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Rescue latent orsubliminal infection
DC/T4 co-culture with compound
14 days 7 daysp24 Ag +
HIV-1
infection 2h
wash step
30 min± ±or
↓↓
¤
↓↓¤
Cell-free virus Compound Compound ¤ T4 cellsT4 cellsCell-associated virus
MO-DCMO-DC PBMC-PHA/IL-2 activated cells
Co-culture model of MO-DC and T4 cells
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Binding/fusionBMS 5TAK 779 42T20 55 140
PMPA 82TMC-120 2 3 2UC781 7 111 52
L 870 812 9 183
Pseudovirus DC/T4 co-culture
+ Ghost-CCR5 + Free HIV + Cell-ass. HIV
EC50 (nM)
848 326 > 10,000
> 10,000 4,500
92RT inhibitors
Integrase Inh
125
1,250
Concluding:All compounds active in PV/GHOST (< 100 nM)
Binding/fusion inhibitors less active with repicative free HIV inactive with cell-associated HIV
Reverse Transc. Inh. very active in all conditions
Integrase Inhibitors intermediate profile
Summary of cell suspension data
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In vivo In vitro
Nature Rev 2006, Lederman MM
Nature Rev 2006, Lederman MM
Dual Chamber model
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0
20
40
60
80
100
120
0 10 100 1000
PRO2000
CS
DS5000
PSS
UC-781
R152929
R153430
R165335 - TMC125
R147681 - TMC120
R151694
R278474 - TMC278
0 10 100
Entry-inhibitors
Non-nucleoside reversetranscriptase inhibitors
Conc. entry-inhibitor (µg/ml)
Conc. NNRTI (nM)
% H
IV p
osit
ive
cult
ures
CONCLUSION: Binding Inhibitors: rather inactiveNNRTI: very active
Effect of Binding Inhibitors and NNRTI against Cell-associated HIV in dual chamber model
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Cervical epithelium(Junction zone)
Migratory cells (DC + T cells)
Explant model
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Cervical epithelium
Migratory cells
Various binding inhibitorsUC781 (NNRTI)
Conclusion: Binding Inhibitors: active, but less against migratory cellsNNRTI: very active, especially against migratory cells
(From R Shattock’s group: J Exp Med 2004 and J Virol 2005)
Inhibition of cell-free infection in explant model
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Issues in further development of microbicides
Incomplete knowlegde of transmission process:
- Cell-free or cell-associated virus ?- Which are the relevant target cells and receptors ?- Role of seminal and cervico-vaginal fluid factors ?- Role of normal vaginal flora/STD and “vaginal practices” ?
Avoiding unwanted side-effects:
- Enhancing infection by epithelial damage or inflammation- Limiting therapeutic options by induction of resistance
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Which in vitro test is suitable and predictive?
Impossible to say until first succesful human clinical trial,
In the mean time:
Use several models reflecting aspects of sexual transmission:e.g. DC and T cells (+ epithelial cells)
Explant model Inclusion of seminal and vaginal fluid factors
In addition: - Ensure activity agains cell-free and cell-associated HIV; - Study optimal drug combinations;
- Thorough evaluation of toxicity; - Study consequences of possible resistance development.
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ACKNOWLEDGEMENTS
Collaborators:
Yven Van Herreweghe; Katty Terrazas; Youssef GaliJo Michiels; Laetitia Aerts; Leo Heyndrickx
Funding
EUROPRISE: sponsored this lecture
EMPRO: European Microbicides Program ANRS: (France)IWT and FWO: Scientific funds of Flemish government DGOS: Belgian Ministry of Development AmfAR: American Foundation for AIDS Research IPM/TIBOTEC CONRAD ITM institutional support