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Candidate Microbicides:
What we can learn from in vitro work
Guido Vanham, MD PhDgvanham@itg.be
Institute of Tropical Medicine, Antwerp
Vaginal HIV transmission
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
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 ?
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
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
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
“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
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
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
In vivo In vitro
Nature Rev 2006, Lederman MM
Nature Rev 2006, Lederman MM
Dual Chamber model
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
Cervical epithelium(Junction zone)
Migratory cells (DC + T cells)
Explant model
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
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
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.
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
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