1. Flutcore meeting
11/4/2016 by
Alex Ramirez PhD

2. 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) .

3. 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

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

5. 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)

6. 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

7. 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

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

9. 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

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

11. 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 +/-

12. 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

13. 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

14. 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

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

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

17. 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