1. BIT225 Reduces the Intracellular HIV-1 Burden Within Monocyte Derived Dendritic Cells, Resulting in Reduced Transfer of Virus to more Permissive CD4 + T Cells J Wilkinson, G Ewart, C Luscombe and M Miller. Biotron Limited, Sydney, Australia Background: Specific viral proteins have ion channel activity (viroporins) led Biotron to develop a library of compounds targeting Vpu. BIT225 has completed both preclinical safety and toxicity studies and a phase I dose escalation study. A phase Ib has recently been completed in HCV + patients with two successive trials planned in HCV and HIV-1 cohorts. In vitro, BIT225 demonstrates good activity in macrophages. (Khoury et al., Antimicrobial Agents and Chemotherapy. 2010) Aims: To extend our findings and determine the anti-HIV-1 efficacy of our lead compound, BIT225, on viral replication in MDDC and determine the potential activity of BIT225 on the inhibition of transfer of HIV-1 from this compartment to more permissive CD4 + T cells when co-cultured. TUPDA103

2. DC and HIV-1 dissemination (2 phase transfer) HIV + iDCmature DCCD4 + T cell Mucosal Tissues Lymph Nodes Maturation & Migration ( `24 h) -CLRs +Adhesion +Activation ‘Trojan Horse’ Turville et al. Blood 103: 2170. 2004

3. BIT225 Inhibits HIV-1 Replication in MDDC TUPDA103 HIV-1 Replication by RT (U/mL) 0 100 200 300 400 500 600 0 12 3 456789101112 13 14 BIT225 20  M DMSO Time (days)  BIT225 resulted in 72% inhibition of HIV-1 replication in infected MDDC  Dose dependent response Methods: Day 6, immature MDDC (CD1a + CD14 - CD4 + DC-SIGN + MR + CD83 - ) infected with HIV BaL and cultured ‘one shot’ dose of ±BIT225 (v DMSO) for 14 days

4. BIT225 Reduces HIV-1 Transfer from MDDC to CD4 + s TUPDA103 Methods: At various time points, infected MDDC ±BIT225 were cultured with PHA activated CD4 + T cells (HIV-1 seronegative donors) at a ratio of 1:4 for 4 days of co-culture with no additional drug added  BIT225 resulted in 89% inhibition of HIV-1 transfer from MDDC to CD4 + s  in cis only, supporting the role of BIT225 as a late stage viral inhibitor

5. In Summary A single dose of BIT225 resulted in reduced:  HIV-1 replication in MDDC (72% inhibition out to day 14)  Transfer to T cells in co-culture (89% inhibition)  in cis only, supporting the role of BIT225 as a late stage viral inhibitor TUPDA103 “BIT225 has the potential to decrease HIV-1 replication in infected dendritic cells. In these infected cells, BIT225 may also assist in reducing the dissemination of HIV-1 to more permissive host cells, as the HIV laden DC traffic from peripheral sites of infection to CD4 + T cell rich lymph nodes”