BBI Biotech Research Laboratories Core B Janet L Lathey, Ph.D. Director Virology/Immunology

Boston Biomedica, Inc. Overview Founded in 1986 Leading supplier of innovative products and services to the life sciences industry Approximately 200 employees with facilities in Massachusetts and Maryland Customers include: –Diagnostics and pharmaceutical manufacturers –Government research and regulatory agencies –Proficiency organizations –End-users of diagnostic test kits 2

BBI Diagnostics Overview ISO13485 certified manufacturer of quality control products used to monitor and measure the accuracy of test results Products include ACCURUN ® Controls, Quality Control Panels, Basematrix, specialty reagents and ACCUCHART™ QC Systems Diagnostic products manufactured from human plasma, serum and cultured virus in a 32,000 sq. foot facility An inventory of approximately 14,000 individual plasma units and specimens 46

Biotech Overview The research and development arm of the Company for Molecular Biology, Virology and Immunology Experienced Scientific Staff consisting of 9 PhD’s and more than 50 scientists Provides a variety of products and services to BBI operating units and other outside customers –Specialty reagents and molecular and cellular biology services –Blood and tissue processing and repository services –Clinical trials for domestic and foreign test kit and device manufacturers Services typically provided under multi-year contracts Services focused in advanced biomedical research areas Frederick, MarylandGaithersburg, Maryland 17

Business Focus Government Contracts Genetic Disease and Cancer Genotyping Clinical Trials Support Repository Operations R&D for Assay Development and Panel Production Technical Support for BBI Diagnostic Products GMP Manufacture of virus, purified antigens and components Molecular Testing and Virus Genotyping Stability Studies Infectivity Studies Research Products and Services Nucleic Acid Extraction and Amplifications Sample processing, storage and tracking Virus isolation and characterization 19

Repository Services Study design support for research and clinical trials Training and documentation for collection sites Blood fractionation, PBMC Isolation and Cryopreservation Specimen accessioning utilizing bar coding and real-time database Sample storage and distribution Shipping guidance and support 24

Molecular Biology Services Extraction of Nucleic Acids –Blood, buccal swabs, tissues HIV Drug Resistance analysis –Viroseq kits from ABI Viral Sequencing and Subtyping Viral Load Assays –Roche COBAS –Roche Amplicor Monitor –RT TaqMan PCR Recombinant DNA and Library Screening and DNA Finger Printing 33

Virology/Immunology Viral Culture, Titration and Inactivation HIV Drug Susceptibility Assays EBV Transformation ELISpot Assays Apoptosis Assays

Virology Services and Molecular Biology Available for Clinical Trials of Anti-retrovirals HIV Antigen Detection and Quantitation HIV Culture, Isolation, and Tropism Titration of Infectious Virus Drug Susceptibility Assays Viral Nucleic Acid Isolation Viral Nucleic Acid Quantitation Viral Sequencing Drug Resistance Testing (Genotyping)

HIV-I Subtypes Collection Subtype# Isolates –A3 –AG3 –B10 –C7 –D3 –AE10 –F5 –G2 –H1 –Group O4 –HIV-21 Our collection includes all members of the NED (NIH-ENVA-DOD) HIV-I Subtypes Reference and Standards Panel All NED Panel isolates were sequenced by the VQA to ensure >98% homology to Panel Seed Stocks. VQA EM Particle Counts for most isolates RNA concentration >10E+08 copies/mL for all isolates GenBank Accession Numbers available Cultured under GMP conditions

Drug Resistant Isolates IsolatesBiotypeResistanceReferences 139-1SI (R5X4) AZT1989 Larder BA, Science 246: 1155 HIV- 174V/MT2 X4ddI,ddC xxxHIV-1 LAI-M184V 1993 Schinaizi RF, Antimicrob Agents Chemother 37:875 3TC N119SI (X4)nevirapine J 302saquinavir1995 Jacobsen H. Virol 206:527

Feed every 3 to 4 DaysHarvest at Day 7,14, 21 and 28 HIV Infectivity Assay Make 10-fold Dilutions of Virus – Add Cells (Cell line or PBMC) Incubate Overnight Centrifuge Cells and Wash 3 Times Incubate for 7-28 Days Measure HIV-1 p24 Antigen by ELISA

Drug Susceptibility Assays Viral isolate is titrated under conditions of susceptibility assay. Decreasing concentrations of drug are diluted with 200 TCID 50 of virus and 200,000 PBMC per well On day 4 drug is replenished On day 7 supernatant is harvested for p24 antigen assay. IC 50 is determined based on reduction in p24 production compared to no drug

Drug Family Drug Identification IC 50 (µg/ml)EC 50 (µg/ml) Therapeutic Index AlkaloidsZS-114>100 e 0.8>125 ZS-147>1001.4>69.9 ZS-156>1000.8>125 TerpenoidsIC IC ,418 SXZ-LN-3>1001.1>91 LignansSXZ-EL SXZ-EL ZS-3>1004.2>23.8 GlycosidesZS-183>1001.0>100 ZS

Anti-retroviral selected with BBI screening is in Clinical Trials Over 5000 plant extracts and derivatives were screened by BBI Biotech and described in over 30 publications. One drug from the terpenoid group was selected for further testing. A Phase I Clinical Trial has been completed with this maturation inhibitor, PA 457. Panacos expects to start a Phase II trial by the end of the year.

Specific Aim (1) Screening potential inhibitors: SP antagonists with potential anti-viral activity will be tested for inhibitory activity against HIV BaL and NL- 43 propagated in PBMC. Cytotoxicity against PBMC will be determined to obtain an in vitro therapeutic index (TI). Compounds with High TI will advance to the next screening level Project 1 Testing antiviral activity In other cell types Remain in Core B To be tested against Primary isolates Project 2 Test effect on monocyte cellular function

Specific Aim (2) Efficacy against primary HIV-1 isolates: Characterize the breadth of inhibitory activity of the compounds against a panel of primary HIV-1 isolates propagated in PBMC. B and non-B subtypes T cell- and monocyte-tropic Anti-retroviral resistant Isolates isolated by Project 1 In presence or absence of inhibitors All data is transferred to Core C with screening data from projects 1 and 2. Complied data will be used to determine best dose of chosen inhibitor

Specific Aim (3) Synergy with anti-retrovirals: To determine if lead compound(s) work in synergy with anti-retrovirals with different modes of action, experiments will be set up with lead compounds in combination with individual or multiple existing anti-retrovirals. R5 and X4 viruses will be used Cytotoxicity of the drug combinations will be determined Results will be transferred to Core C for addition to the data base. Compiled data will used to anticipate drug interactions in future clinical trials

Specific Aim (4) Loss of susceptibility: HIV will be continuously cultured with low doses of lead compound. The virus will be periodically tested for changes in the level of susceptibility to the lead compound. Compound(s) which make it through the above screening procedures and are targeted for clinical trials will be tested in long term culture with a susceptible strain of HIV. Cell cultures will be maintained with low doses of compound with and without virus. The virus will be tested in a drug susceptibility assay once a month using “normal cells” and cells that had been cultured with the same concentration of compound as virus. If there is a consistent shift of the inhibitory concentration with normal cells the virus will be sent to Project 1. If there is a shift with treated cells and not “normal cells” and there is also a shift with parent (untreated) virus in treated cells, the cells with both viruses will be sent to Project 2 to look for changes in NK-1R

Specific Aim (5) Efficacy against SIV: SIV strain targeted for use in the macaque mode will be tested in PBMC against lead compounds for drug susceptibility. Viruses will also be isolated from infected macaques and tested for drug susceptibility in conjunction with parent virus used for initial infection. Initial testing results will be forwarded to Project 3 to facilitate decisions about drug concentrations to be used for testing. Results of these tests will be sent back to Project 3 via Core C to be correlated with clinical results.

Specific Aim (6) Sensitivity of patient isolates: To determine the susceptibility of isolates from patients participating in the clinical trial; viruses, isolated from individual patients, will be tested in vitro against the SP antagonist. This will be performed pre and post drug administration. Data will be sent to Core C for correlation with clinical trial results (Project 4). Viruses which demonstrate a change in susceptibility with increasing treatment during the trial will be evaluated to determine if viral receptor interactions have changed. Viruses with changes in receptor interactions will be sequenced by Core B