Flutcore meeting 11/4/2016 by Alex Ramirez PhD

Last meeting VLP vaccine candidates chosen HA2.3, 3M2 coded as VLP1 LAH3, K1 coded as VLP 2 - These candidates were immunogenic, protective and universal (cross-reactive) .

Since last meeting: Testing of 3P material LAH3,K1 from before Christmas 3P pellet prepped by iQur 2 immunisations (ip/sc) with Alhydrogel only 6week total/ 12 days post final boost

Assay development Need for assays to detect/characterise VLP for various applications: -IPC -QC / EPC -Product stability - IVRP 2 assays developed

Capture ELISA- based on Gardasil IVRP - Detects presence of VLP Detects presence of protective antigen Can be quantitative of VLP, within a protein mixture. Can be optimised to favour VLP detection over monomer detection. Anti- HA stalk LAH (not commercially available) VLP2 Anti- HB core# (10E11 Abcam or similar)

Validation of in- house anti-HB core Ab: AX-Core. -Validated for Elisa, WB and IP (not shown) applications VLP2 VLP2 HBc monomeric 50kDa 15kDa Ctrl 10E11 Abcam AX-Core Ctrl VLP2 Monomeric HBcore 1 1:10

Validation of in- house anti-LAH3 Ab: AX-LAH Also validated for IP and for HA3 WB (not shown) Hemagglutinin WB VLP2 Dot Blot 50kDa HA3 (H3N2) HBc VLP2 HA3 ctrl -not yet tested for VN or protection

Capture ELISA- for VLP 2 -linear range 0.5 – 5 ug/ml of VLP Different stopping times AX-LAH -HRP VLP2 AX-Core

HPLC- SEC assay -IPC -QC / EPC -Product stability - Agilent HPLC 1260 infinity 20mMTris pH8.4 5mM EDTA SEPAX column: SRT SEC-1000, 5uM, 1000A silica matrix

SEC Gel Filtration Biorad standards Overlay with LAH3,K1 280nm trace/ 0.3ml/min 151-1901 Myoglobin 17 VLP 2 prep Vitamin B12 1.35 SEC marker g- globulin 158 Ovalbumin 44 Thyroglobulin 670

Calibration curve (iQur)- SEC HPLC -0.3ml/min 20 min run setting -Protein complexes in the 5-7 MDa mw range should have retention times of 8min +/-

Known VLP runs (purified preps) 280nm trace/ 0.3ml/min Known VLP runs (purified preps) 0min 20min 8.2 HBc wt (commercial Source ) BL21 empty tandem 7.7 7.7 3M2, K1 Riga 8.4 VLP2

HPLC traces of Clarified lysate 8.5 8.0 8.5 8.8 We have now developed a standardised method for assembled VLP identification using HPLC This relies on SEC retention times

Clarified Lysate LAH3,K1 (pH 8.5) 20min 0min HPLC trace 280nm OD at 0.3 ml/min LAH3,K1 Lysate Fr8 HA2.3 , 3Mctrl 50kDa Lysate shows core positive material is in fractions around time 8min (on 20min total run) These fractions are core and LAH3 positive by dot blot VLP are visible by EM Molecular weight calibration suggests 8mins corresponds to 5MDa (this is correct for an assembled VLP) SEC-HPLC is a viable assay for VLP formation AX-core

? Prime (buffer only) 8 Lysate (clarified) TFF output Purification Stages CL4 B output AEX fr 15 0min 20min

Current - S1000 final VLP1 and VLP 2 preps pH8.4 (Cmut) VLP2

Summary 2 Antibodies developed : AX-Core and AX LAH 2 more Ab underway : AX- HA2.3 and AX- M2 (clone selection stage) Capture ELISA assay developed HPLC SEC assay developed