Virology Platform

Research Service Division http://rsd.wuxiapptec.com WuXi AppTec Virus Platforms ● Animal models ◦ Hydrodynamic injection (HDI) mouse model AAV/HBV mouse model HBV ◦ HBV ◦ Humanized FRG mouse model HSV HCV Duck/DHBV model HCMV HDV ◦ Transgenic mouse model IFV ◦ EBV in vitro ◦ WHV/woodchuck model HPIV VZV screening and profiling ● Immunological assays supporting animal models HRV ◦ Isolation of immune cells: lymphocytes from mouse spleen, liver and HHV-6 in vivo efficacy lymph nodes, PBMC from blood HIV clinical virology RSV ◦ FACS: surface and intracellular markers ELISPOT EV71 HAdV ◦ ◦ Cytokines by Luminex, ELISA, MSD and RT-qPCR CV Zika RSVHCoV Zika DENV Cpd A PHH post HBV infection (h) 100 0 6 24 32 48 56 72 120 144 240 DES19

rcDNA

Antiviral: from Assay Setup to Clinical Trial Support 50 dslDNA

cccDNA % inhibition Develop bioassays 3.5 kb ● 0 ◦ Biochemical assays 0 1 2 3 4 2.1/2.4kb ◦ Cell-based assays Log cpd conc. (nM) ◦ Virological assays

10 HBV DNA in serum ● HTS 9

◦ Drug combination ● Support lead ID/Op, SAR, PCC 8 ◦ Serum shift ◦ Compound screen ◦ Genotypic spectrum 7 ◦ Characterization ◦ Pan-virus panel log (HBV DNA copies/ml) ◦ MOA studies ◦ Cross resistance 6 ◦ De novo selection of drug- resistant virus 5 0 10 20 30 40 50 60 70 80 90 100 ◦ In vivo and ex vivo efficacy Support clinical trials Days post AAV/HBV infection ● ◦ Customer designed studies ◦ Sequencing ◦ IND package ◦ Genotyping ◦ VL Other HBV assays ◦ Phenotyping ● cccDNA: Southern blot and qPCR ◦ Host factors ● HBsAg, HBcAg and HBeAg, anti-HBs and anti-HBc: ELISA and IHC ● Intracellular HBV DNA replicative intermediates: Southern blot ● HBV transcripts: Northern blot and RT-qPCR CAP certificate ● Encapsidated DNA and RNA ● Extracellular HBV virion: Immunoprecipitation and qPCR LIVER-SPECIFIC VIRUSES ● Cell-based HBV RNase H assay ● and core contents ● Serum HBV RNA and HBcrAg B virus (HBV): key service capabilities ● Cytokines, IFNs and ISGs: Luminex, ELISA and RT-qPCR ● Drug screening using HBV producing cell lines ● Immune cells: isolation, FACS and ELISPOT ◦ HepG2.2.15, DE19, and DES19 cells with qPCR, Southern blot, Northern blot, ELISA, cccDNA, encapsidated pgRNA and DNA ● Apoptosis: FACS and TUNEL ● HBV infectious assays ● Core protein expression: Western blot and immunostaining ◦ Ex vivo primary human hepatocyte (PHH) culture system ● Mechanism of action (MOA) studies ◦ HepG2-NTCP cells ● Production of HBV ● Reporter assays: hTLR, THP1-Blue™ ISG, TNF-Induced NF-κB-luciferase cell lines GLS4 lamivudine ● Core protein expression and capsid assembly quenching system ● Compound profiling

100 nM Drug combination, serum shift, 250 nM 1000 nM DMSO ◦ 1000 nM ◦ Clinical isolates and genotypes (up to 5 constructs for each of A to H) ● Drug-resistant mutants HBV capsid ◦ Nuc (16 constructs) and CpAM (16 constructs) resistant mutants by transient transfection (qPCR) Capsid-associated ◦ Construction of drug-resistant mutants, assessment of fitness and drug DNA susceptibility Capsid-associated Western Blot ● Clinical virology RNA ◦ Full-length HBV genome sequencing (Sanger and Deep Sequencing), Cpd A genotyping, VL Core protein HBcAg ◦ Phenotyping (full-length, , capsid shuttle vectors) ◦ New HBV markers: serum HBV RNA, HBcrAg, HBV RNA sequencing β-actin β-actin

Hepatitis C virus (HCV): key service capabilities 100

● Screening: stable and transient replicons, and HCVcc with different 75 endpoints 50 ● Profiling ◦ Drug resistance: cross resistance, de novo resistance selection % inhibition 25 ◦ Genotypic spectrum 0 ◦ Drug combination -3 -2 -1 0 1 2 ◦ Serum shift Log[Compound, • M] ◦ Colony formation ◦ RSV: Long strain ◦ RSV F protein mediated cell Cells: HEp-2 fusion assay ● Clinical support ◦ ◦ Compound treatment: 5 days ◦ VL (COBAS® AmpliPrep and TaqMan 48 systems) ◦ Endpoint: CPE ◦ Genotyping ◦ Phenotyping (chimera, SDM, fitness, drug susceptibility) Human Coronavirus (HCoV) ● Biochemistry: viral enzymes ● HCoV 229E and OC43, cell-based CPE and qPCR assays SARS-CoV-2 Mpro enzymatic assay Hepatitis D virus (HDV) ● ● Other assays are under development A cell-based infectious assay ● Human rhinovirus (HRV) RESPIRATORY VIRUSES ● HRV 1B (Subtype A) and HRV 14 (Subtype B) strains, cell-based CPE assay ● Mouse nasal infection model with viral load in lungs, cytokines and H&E stainin virus (IFV) ● Screening/profiling Human parainfluenza virus (HPIV) ◦ A mini panel of H1N1, H3N2 and B strains ● HPIV 3, cell-based CPE assay ◦ H7N9, H5N1 (subcontract lab) ● HPIV GFP reporter assay under development ◦ Cell-based CPE, plaque and qPCR ◦ De novo selection of drug resistance ◦ Serum shift and drug combination Human adenovirus Oseltamivir, VX-787 and Baloxavir resistant viruses ◦ ● AdV 14 cell-based CPE assay In vivo model ● ● RCA assay ◦ Mouse nasal infection with mortality, body weight and VL ◦ Ferret nasal infection with body weight and viral load ● Biochemistry Porcine reproductive and respiratory syndrome ◦ Neuraminidase, hemagglutinin, polymerase virus (PRRSV) ● Purification of and replication complex from infected hen ● Cell-based CPE assay eggs

100

80 HERPES VIRUSES

60 Uninfected+vehicle H1N1+vehicle 40 Oselt 1 mpk Oselt 10 mpk Herpes viruses Oselt 30 mpk Mouse Mouse survival (%) 20 ● virus (HSV) 0 ◦ HSV-1 and HSV-2, cell-based CPE assays 0 2 4 6 8 10 12 14 In vivo model: (1) mouse intranasal and vaginal infection lethal model, 2 Day post infection ◦ mortality, body weight and viral load (2) Mouse ocular latent infection 0 and activation model Ex vivo model: isolation and cultivation of latently infected ganglia -2 Uninfected+vehicle ◦ H1N1+vehicle neurons -4 Oselt 1 mpk Human cytomegalovirus (HCMV) Oselt 10 mpk ● -6 Oselt 30 mpk ◦ Cell-based CPE and reporter replication assay Body weight (g) change -8 In vivo model: NOD SCID mouse, sponge virus implant with VL by plaque 0 2 4 6 8 10 12 14 ◦ day post infection assay ◦ H1N1: i.n. inoculation ● Epstein-Barr virus (EBV) ◦ Oseltamivir: po, bid, 1st dose at 4 h prior to infection, 7 days ◦ Cell-based qPCR assay ● Human herpes virus 6 (HHV-6) Respiratory syncytial virus (RSV) ◦ Cell-based qPCR assay Viral infection assays ● ● Varicella-zoster virus (VZV) RSV A: Long and A2 ◦ ◦ Cell-based CPE and qPCR assays ◦ RSV B: B9320 and B18537 Gelfoam implants from an NOD SCID ◦ Clinical isolates: RSV A and B mouse (9 days post implantation) ◦ CPE, plaque, ELISA Profiling: drug resistance, genotypic spectrum, drug combination T D F E C 5 0 1 6 .9 7 n M ● HIV-1 1 2 5 ● MOA studies Viruses: HIV-1 IIIB or BaL 1 0 0 %

● 7 5 n o

De novo selection of virus resistance i t

◦ i 5 0

Reference: AZT (Retrovir) or TDF b

● i h

F protein-based cell fusion assay n 2 5 ◦ (Viread) I ● Mouse nasal infection (A2) model with VL in lung by the plaque assay 0 ● MT-4 cells, CPE assay with CellTiter -2 5 ● Cotton rat nasal infection model with VL in lung by the plaque assay Glo -2 -1 0 1 2 3 lo g 1 0 [c p d c o n c ., n M ] Hand-foot-mouth disease viruses 3 OV Validation ● Enterovirus 71 (EV71) ● In vitro efficacy ● In vivo efficacy ◦ Cell-based CPE assay ◦ Identity assay: qPCR, WB, Elisa ◦ CDX/PDX/syngeneic animal Cyto toxicity assay: CCK8, models for efficacy validation ◦ EV71 Shenzhen/120F1/09 ◦ crystal violet, co-culture with ◦ TME/immune response: IHC, ◦ EV71 MAV-W PBMC or splenocyte FACS, ELISpot ◦ EV71 mouse lethal and VL qPCR model ◦ Immune assay: ELISA, FACS Transgene mouse model for EV71 clinical ◦ Infected Uninfected strain (under validation) 4 Assay Capability ● Coxsackie virus (CV) ◦ Cell-based CPE assay ● Virus titration: TCID50, plaque ● Immunogenicity analysis for A16 SZ05 assay transgene: FACS, ELISA, WB, CV A16 G08-M ● Virus shedding or distribution: qPCR B3 Nancy qPCR, TCID50, plaque assay ● in tumor ◦ A16 mouse lethal model and VL qPCR model ● Neutralizing biopsies: IHC detection: CPE, ELISA Other viruses ● Zika virus (PRVABC59) ● Dengue virus Vaccine evaluation ◦ Type 2 cell-based CPE assay ◦ Cell-based CPE and plaque ● In vitro assays assays ● Human adenovirus Generation and production of vaccines, such as inactivated, attenuated, AdV 14 cell-based CPE assay ◦ ◦ AG129 mouse model ◦ subunit (proteins or VLPs) and viral vector based vaccines (subcontract lab) RCA assay ◦ ◦ Titration of neutralization Ab in serum Immunological assays ◦ Detection of immunoglobulins ◦ Assessment of cytokines ● PBMC: isolation of human, mouse, rat, dog or monkey PBMCs ◦ Virus distribution and PK ● Lymphocytes: isolation of lymphocytes from liver, spleen and IHC/HE lymphonodes and other organs/tissues ◦ ● In vivo models ● Analysis ◦ IFV mouse and ferret infection models ◦ Cytokines/chemokines, IFN and ISGs by Luminex, ELISA, RT-qPCR ◦ RSV mouse and cotton rat infection models ◦ FACS ◦ HFMD neonatal mouse infection models (EV71 and CA16) ◦ Intracellular staining (ICS) ◦ HSV-1 mouse infection model ◦ T and B cell ELISPOT ◦ Dengue mouse infection mode (subcontract) ◦ Check points ◦ Zika mouse infection model (subcontract) ◦ Immunohistochemical staining (IHC) Viral vector (VV) capabilities Clinical virology ● Various viral vectors for cloning target genes ● CAP-certified clinical virology support (HBV, HCV, RSV, IFV) ● Preparation of endo-free plasmid DNA in various scales ◦ Viral load, qPCR /RT-qPCR, ddPCR ● Package and production of recombinant viruses ◦ Sequencing ● Purification of recombinant viruses with density gradient based ◦ Genotyping ultracentrifugation ● Phenotyping ● Titration of virus using qPCR, plaque assay, TCID50 and commercial kits ◦ HBV full-length, pol and capsid shuttle vectors HCV replicon shuttle vectors ● Analysis of protein impurities of the final products ◦ ◦ Drug susceptibility and fitness ● Determination of the bioactivity of the final recombinant virus products ● HBV RNA Assessment of empty capsid by EM ● ● HBcrAg Animal PK/PD studies ● ● Viral antigens and antibodies ● PK/PD sample analysis: virology assays, ELISpot, FACS, ELISA, MSD, Cytokines, IFN and ISGs Luminex, RNASeq, qPCR, etc. ● ● Others ◦ Oncolytic viruses Viral vector platform: oncolytic viruses ◦ CAR-T Host markers: IL28B genotyping, MET exon 14 skipping, etc. 1 Vector Modification ◦ ● Ad-Easy/BAC system/CRISPR ● Modifications ◦ pAdeasy1 ◦ E1/E3//ICP34.5 gene deletion ◦ pShuttle ◦ Fiber modification for chimera ◦ pCC1BAC or cancer receptors ◦ pBAD-I-SceI ◦ Insertion of siRNA or immune ◦ Lenti-crispr_v2 genes

2 Virus Production ● Virus packaging and ● Virus production and identification purification ◦ PCR/qPCR ◦ Surcrose/iodixanol/ Enzyme digestion/RFLP CsCl density gradient ◦ ◦ qPCR system in Molecular ◦ Fluorescence detection with ultracentrifugation Elispot ◦ Titration Testing Lab ◦ limited dilution with plaque

Contact Declan Ryan (US) Dave Madge (Europe) Longji Xu (China) Vice President, Business Development Vice President, Business Development Director, Business Development [email protected] [email protected] [email protected]