iQur Limited TSB Q8 Meeting 2 nd September 2009

Agenda MW: project update & meeting schedule AET: Leeds purification MW: in vivo data LB: new constructs/EM MS: yeast expression LB: Arecor EM BD: stability studies

Dates for meetings Final meeting: Week beginning 30 th November 2009 UCL

Outstanding work (Q7) Arecor to develop stability buffer –In progress iQur to carry our pre and post in vivo study on this material (is there enough?) –Awaiting material Mologic to express and purify yeast derived material –In progress iQur to carry out in vivo study using this material –Awaiting material

Q8 TSB Meeting In vivo update

UCL14 Dual insert construct of sAg and GFP Formulated +/- adjuvant (alum) Engerix control at molar equivalent level to sAg insert Three vaccinations at weekly intervals Sampling at days +7, 21 and 28

UCL14 Core ELISA (d28) Core response maintained and strong No IgM at day 28 but was detected at day 7

UCL14 eGFP ELISA (d28) GFP response best when adjuvant present Dual and single Inserts have a very similar profile

UCL14 Surface ELISA (d28) No detectable surface antigen response Control working well Neither IgM or IgG

UCL15 Repeat of dual insert construct of sAg and GFP (sAg and GFP alone controls) Formulated in alum Engerix control at molar equivalent level to sAg insert 1,2 or 3 vaccinations at weekly intervals Sampling at days +7, +14 and +21

UCL 15 Core ELISA (d 7)

UCL 15 GFP ELISA (d 7)

UCL 15 sAg ELISA (d 7)

UCL15: ELISA Core response titrates for 1-3 doses GFP response titrates for 1-3 doses Plant derived GFP-VLP is positive Single sAg insert is weakly positive (day 7) but dual insert is not.

Flow cytometric analysis of VLPs Provides a rapid analysis of VLP structure Assay is simple enough to be used in batch process/release Provides data relating to the interaction of inserted epitopes Fully quantitative

Assay design GFP antigen detected in FL1 channel or use FITC conjugated antibody to insert 2 VLP immobilised onto protein G beads by anti-core PerCP conjugated antibody to insert 1 (or SA-PerCP)

Steric hindrance sAg single insert Antigen detected in upper quadrants sAg/GFP dual insert sAg is no longer detectable but beads do fluorescence green proving VLP has been captured Single sAg insertIsotype control Dual sAg GFP

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