1. Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia

2. Further evaluation of HA stalk constructs for diagnostic and vaccine purposes

3. HA undergoes structural changes at different pH Pre-fusion (neutral pH) Post-fusion (low pH) Xu and Wilson, 2011 Current attempts to make vaccines try to utilize pre-fusion conformation

4. Evaluation of HA stalk constructs HA2.3 trimer resembles HA post-fusion conformation at low pH No crystals obtained at neutral pH NMR data at neutral pH suggest high flexibility native HA HA2.3

5. Evaluation of HA stalk constructs A portion of HA2.3 trimer sent to LUX colleagues Results: N-terminal non-structured part should be deleted? HA2.3 trimer is well suitable for ELISA assays… …but probably not good enough for protection

6. Evaluation of HA stalk constructs HA2.3 = 72 aa LAH = 57 aa Crystals obtained at different pH range… …but still in post-fusion form Easy to purify Forms stable trimer

7. Evaluation of HA stalk constructs Possible solutions: Pre-fusion stabilizing mutations Exposure on VLPs

8. Evaluation of HA stalk constructs HA stalk pre-fusion structure Distance between C-termini of monomers about 12-13Å

9. Evaluation of HA stalk constructs Structure of small RNA phages – phiCb5 example Distance between N-termini of monomers about 13-16Å – similar to distances between monomers in HA stalk

10. Evaluation of HA stalk constructs Is it possible to fuse HA stalk C-terminus to N-terminus of small RNA phages and get particles? ? In theory – yes… Also, the pre-fusion conformation might get stabilized In practice, many variants should be tried…

11. Incorporation of HA stalk fragments into VLPs LAH PP7 Small RNA phages tested: Qb, GA, AP205, PP7 Soluble product obtained in case of some PP7 and AP205 constructs The most promising up to now:

12. Incorporation of HA stalk fragments into VLPs Purification of LAH-PP7 protein Immunogenicity to be tested…