candidate microbicides: what we can learn from in vitro work

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