Fig 1: HIV-1/HCV and HIV-1/HBV Co-Infection/Co-Culture Systems

Philippe A. Gallay1, Udayan Chatterji1, Michael Bobardt1, Daren Ure2, Dan Trepanier2, Robert Foster2 and Cosme Ordonez2

1Department of Immunology & Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA. 2Ciclofilin Pharmaceuticals

BACKGROUND & AIMS

HCV/HBV-related liver disease is the main cause of morbidity and mortality of HIV-1 patients co-infected with HCV and/or HBV. Despite the recent advent of anti-HCV DAAs, the treatment of HCV/HBV/HIV-1 co-infected patients remains a challenge, as these patients are less responsive to treatment, have higher rates of re-infection, and develop liver fibrosis, cirrhosis and liver cancer more often than mono-infected patients. In this study, we used a novel in vitro co-infection model to demonstrate that CPI-431-32, a novel cyclophilin A (CypA) inhibitor, simultaneously blocks replication of HCV, HBV and HIV-1 in human cells.

RESULTS

Fig 3: In HIV-1/HBV Co-Infection Model, CPI-431-32 Simultaneously Inhibits HIV-1 and HBV

Fig 4: Dual Block of HBV InfectionCONCLUSIONS

ACKNOWLEDGEMENTS

METHODS

Using a unique and novel in vitro co-infection model, we examined whether CPI-431-32 interferes i) with HCV RNA synthesis of isolated replication complexes using a quantitative replicase assay, ii) with early steps of viral replication of HIV-1 primary isolates, and iii) HBV virus entry (NTCP binding) and viral replication. We also tested by ELISA the CPI-431-32 inhibition of the interaction between CyPA and the viral proteins NS5A and p24gag of HCV and HIV-1 viruses, respectively, to explore the mechanism of action of this drug.

The unique broad-spectrum of antiviral activity shown by CPI-431-32 might be related to the role of CypA in viral infection. CPI-431-32 prevents binding of CypA, a protein-folding enzyme to viral proteins such as HCV NS5A and HIV p24gag, which are critical for HCV and HIV-1 replication. We hypothesize that the inhibition of CypA by CPI-431-32 is responsible for its broad-spectrum antiviral activity by preventing activation of specific viral proteins required for viral replication. Overall, this study suggests that CPI-431-32, a novel CypA inhibitor, could potentially become a candidate medicine for the treatment of HIV-1 patients co-infected with HCV and/or HBV.

We thank F. Chisari for the Huh-7.5.1 cells, T. Pietschmann, T. Wakita and R. Bartenschlager for the Luc-JFH-1 plasmid, and C. Seeger for HepAD38 cells . This work was supported by the U.S. Public Health Service grant no. AI087746 from the National Institute of Allergy and Infectious Diseases (NIAID) and a research grant from the Canadian Institutes of Health Research (CIHR).

Novel Cyclophilin Inhibitor CPI-431-32 Shows Broad Spectrum Antiviral Activity by Blocking Replication of HCV, HBV and HIV-1 Viruses

RESULTS

We found that CPI-431-32 blocked viral replication of HCV, HBV and HIV-1. CPI-431-32 was more effective than alisporivir (ALV) (another CyPA inhibitor) inhibiting replication of each of these three viruses. We also demonstrated that CPI-431-32 blocks nuclear import of HIV-1 virus. CPI-431-32 blocked binding of CyPA to HCV NS5A and HIV-1 p24gag more efficiently than ALV, and was more effective than ALV against established resistant-variants of HIV-1. No other antiviral agent tested in our assays was able to show such a broad-spectrum of antiviral activity.

Fig 2: The Cyclophilin Inhibitor CPI-431-32, but not the HCV NS5A Inhibitor Daclatasvir nor the HIV-1 Protease Inhibitor Nelfinavir, Simultaneously Inhibits HCV/HIV-1 Viral Replication in a Co-Infection Model

HCV JFH-1-infected hepatoma Huh7.5.1 cells and HIV-1 JR-CSF-infected PBMCs (duplicates) were incubated together for 3 days and then exposed to a single dose (2 µM) of the cyclophilin inhibitor CPI-431-32, the HCV NS5A inhibitor daclatasvir, the HIV-1 protease inhibitor Nelfinavir or drug combinations. HCV and HIV-1 replications were quantified by HCV core and HIV-1 capsid/p24 ELISA. Data are representative of 2 independent experiments.

HIV

HBV

Fig 5. CPI-431-32 Blocks Formation of HBV cccDNA

HIV-1 JR-CSF-infected PBMCs and HBV AD38-infected NTCP-Huh 7 cells (duplicates) were exposed to a single dose (2 µM) of CPI-431-32 and then incubated together for 3 days.

Viral replication was quantified by HIV-1 capsid/p24 ELISA and HBV HBeAg ELISA. Data are representative of 2 independent experiments.

HepaRG cells (duplicates) were exposed to HBV AD38 together with ALV or CPI-431-32 and viral replication was quantified by HBV HBeAg ELISA at day 3.

NTCP-Huh7 cells (duplicates) were incubated with HBV preS1-FITC peptide together with ALV or CPI-431-32. HBV preS1 binding to cell surface NTCP was quantified by FACS.

Fig 6. Mechanism of Anti-HBV Effect for CPI-431-32

Huh7, HepG2 or NTCP-Huh7 cells were transfected with HBV in the presence of DMSO, ALV (2 µM) or CIP-431-32 (2 µM).

Three days post-transfection, amounts of HBV cccDNA were quantified by PCR as described previously (Gao and Hu, J. Virol. 2007).