C1 rHA-TMD from Dyadic Immunogenicity study in mice Test - PowerPoint PPT Presentation

C1 rHA-TMD from Dyadic Immunogenicity study in mice Test MLE-PT-RFA-15002-Ms Mise à jour du 14 sept 2015 CONFIDENTIAL | 1

Three questions 1. Does the rHA-TMD induced clinical signs in mice? 2. Is the C1 rHA-TMD immunogenic and how does it performed compared to the baculo-rHA? 3. Is there an adjuvant effect due to the C1 matrix at high dose? CONFIDENTIAL | 2

Evaluation of C1 rHA-TMD from Dyadic Tested Products and characterization Test MLE-PT-RFA-15002-Ms Denomination Batch Number Protein content / HA content Endotoxin Purity (IU/mL – BRD) Recombinant HA from the 1869 BCA (sp): 791 µg/mL 12 80% A/H1N1/New µBradford (sp): 627 µg/mL Caledonia/20/99 strain SRID: 129 µg/mL produced in Myceliophthora Titre UHA: 1280/50µL thermophila (C1) TMB (Dyadic , Engen) Recombinant HA from the 1367-057 BCA (Protein Sciences): 686 µg/mL <10 >90% A/H1N1/New µBradford (sp): 391 µg/mL Caledonia/20/99 strain SRID: 200 µg/mL produced in baculovirus Titre UHA: 32000/50µL (Protein Sciences) C1 mock1 TMD-rHA (Dyadic, 1860 BCA (sp): 970 µg/mL 79 N/A Engen): AEX fractions 4-10 µBradford (sp): 970 µg/mL that eluted before the SRID: ND fractions where HA Titre UHA: 16 normally elutes off the column C1 mock2 TMD-rHA (Dyadic, 1860 BCA (sp):1276 µg/mL 2452 N/A Engen): AEX fractions 11-15 The most µBradford (sp): 1650 µg/mL where the HA normally representative C1 SRID: ND elutes off (same salt mock Titre UHA: 32/50µL concentrations that HA normally elutes off) CONFIDENTIAL | 3

Evaluation of C1 rHA-TMD from Dyadic Biochemical characterization of the two rHAs Test MLE-PT-RFA-15002-Ms Similar oligomeric state between both rHAs 2 1 1 2 HA SDS-PAGE TEM Negative staining Native PAGE 1 C1 rHA TMD 1869 2 Baculovirus rHA PS lot 1367-057 1 N-glycosylation profile of HA by mass spectrometry 2 CONFIDENTIAL | 4

Immunogenicity evaluation of rHA Dyadic (88 mice) Study design - Test MLE-PT-RFA-15002-Ms ● Balb/C ByJ mice - 9 weeks-old - 2 IM immunizations (D0 & D28) under IM 100µl (50µl /thigh): total 88 mice immunizations ● D0 D28 D49 D27 Products  3-Fold Escalating doses of Protein Sciences-rHA : 1 - 3.33 - 10 - 15/06/2015 16/06/2015 07/07/2015 19/05/2015 (W25) 30 µg (8 mice/group=32 mice)  (W28) (W21)   3-Fold Escalating doses of C1-rHA: 1 - 3.33 - 10 - 30 µg (8 mice/group=32 mice)  Negative control groups • HI on D27 and D49 serum samples ● PBS (5 mice) ● C1 mock preparation 1: 30 µg (5 mice) ● C1 mock preparation 2 : 30 µg (2 mice) Statistical analysis ● First: Dose-effect establishment using a model ● of covariance (dose factor after log10 Products transformation as continue variable).  “Spiked” group: 10 µg of Protein Sciences- rHA diluted in 30 µg of Comparison by analysis of variances of HI responses induced by the two rHAs post-1 and C1 mock preparation 1 (8 mice) post-2 injections (IM)  “Spiked” group: 10 µg of Protein Sciences- rHA diluted in 30 µg of ● Second: evaluation of the adjuvant-effect by C1 mock preparation 2 (4 mice) both C1 mock preparations on the Protein Sciences-rHA CONFIDENTIAL | 5 | 5

Question1: Does the rHA- TMD induced clinical signs in in mice? CONFIDENTIAL | 6

Clinical follow-up results- Test MLE-PT-RFA-15002-Ms ● No weight loss (as per weigh measurement between D0 and D28) ● No clinical signs effect during the all experiment (visual observations every day) CONFIDENTIAL | 7

Question 2: is the C1 rHA- TMD immunogenic and how does it performed compared to the baculo-rHA? CONFIDENTIAL | 8

Immunogenicity evaluation of C1 rHA-TMD Results: A/NC/20/99 HI specific response post-1 IM (D27) A/New Caledonia/20/99 (H1N1) HI response after 1 IM immunization in rHA-TMD rHA Baculovirus Negative Controls mice: Myceliophtora (Protein Sciences) Thermophila C1 C1 C1 C1 mock1 mock2 PBS (Dyadic - Engen) mock1 mock2 spiked spiked HI titer against A/New Caledonia/20/99 (H1N1) virus Statistical analysis was not possible 30µg 30µg Not statistically analyzed due to too much non responders 1000 C1-rHA TMD > Baculo-rHA Although: ● Clear evidence of the superiority of 123 C1-rHA TMD over baculo-rHA (cRBC) (number of responders and mean 100 titers) 52 40 40 18 13 Percent of responders 11 10 10 10 6 Dose of rHA 5 5 5 Expression 5 1µg 3.33µg 10µg 30µg system 1 3.33 10 30 1 3.33 10 30 10 10 C1 50% ( 4/8 ) 57.1% ( 4/7 ) 100% ( 8/8 ) 100% ( 8/8 ) µg of A/New Caledonia/20/99 rHA (µBradford dosage) 62.5% ( 5/8 ) 12% ( 1/8 ) 50% ( 4/8 ) 75% ( 6 /8 ) Baculo ● CONFIDENTIAL | 9

Immunogenicity evaluation of C1 rHA-TMD Results: A/NC/20/99 HI specific response post-2 IM (D49) Significant difference : C1-rHA >baculo-rHA A/New Caledonia/20/99 (H1N1) rHA-TMD rHA Baculovirus Negative Controls Myceliophtora (Protein Sciences) Thermophila C1 C1 C1 C1 mock1 mock2 PBS (Dyadic - Engen) mock1 mock2 spiked spiked HI titer against A/New Caledonia/20/99 (H1N1) virus 830 30µg 30µg 1000 640 453 415 381 108 104 123 (cRBC) 95 100 HI response after 2 IM immunizations in 44 mice: ● Significant 2-fold difference in favor of C1-rHA TMD (p-value: 0.006) 10 5 5 5 5 ● CONFIDENTIAL 1 3.33 10 30 1 3.33 10 30 10 10 µg of A/New Caledonia/20/99 rHA (µBradford dosage) | 10

Question 3: is there an adjuvant-effect due to the C1 matrix at high dose? ● CONFIDENTIAL | 11

Evaluation of the matrix effet of C1 rHA-TMD at high dose Results: A/NC/20/99 HI specific response post-1 IM (D27) Not statistically analyzed Spiked groups baculo-rHA Negative Controls baculo-rHA (10 µg) in (10µg) C1 mock1 C1 mock2 C1 mock1 C1 mock2 PBS (30µg) (30µg) (30µg) (30µg) HI titer against A/New Caledonia/20/99 (H1N1) virus 1000 Mock2 most representative Statistical analysis was not possible C1 mock due to too much non responders C1 C1 mock2 mock1 spiked (cRBC) ● spiked Both C1-mock1 and C1-mock 2 100 spiked groups (30µg) showed higher 52 responses than those measured 40 with 10 µg of baculo-rHA alone 10 10 5 5 5 5 ● CONFIDENTIAL | 12

Evaluation of the matrix effet of C1 rHA-TMD at high dose Results: A/NC/20/99 HI specific response post-2 IM (D49) Significant difference : Adjuvant-effect with both C1-mock preparations Mock2 most representative C1 mock Spiked groups baculo-rHA Negative Controls baculo-rHA (10 µg) in (10µg) C1 mock1 C1 mock2 C1 mock1 C1 mock2 PBS (30µg) (30µg) (30µg) (30µg) HI response after 2 IM immunizations in HI titer against A/New Caledonia/20/99 (H1N1) virus mice: 1000 640 ● Significant adjuvant-effet with both 381 C1-mock preparations (30µg) on the baculo rHA (10µg) 123 (cRBC) ● with C1-mock 1 (p-value: 0.0005) 100 ● with C1 mock-2 (p-value: 0.0439) 10 5 5 5 5 ● CONFIDENTIAL | 13

Conclusions 1. The full length recombinant HA from the A/H1N1/New Caledonia/20/99 strain produced in Myceliophthora thermophila (C1 fungi) did not induce any clinical signs in mice 2. The full length recombinant HA from the A/H1N1/New Caledonia/20/99 strain produced in Myceliophthora thermophila (Dyadic , Engen) is at least as immunogenic as the baculovirus-rHA (Protein Sciences) in mice ● C1-rHA batch 1869 > baculo-rHA batch 1367-057 (for those batches and as per µBradford dosage evaluation) 3. Both C1-mock preparations have an adjuvant-effect at high dose (30µg) on the response induced by 10µg of rHA from Protein Sciences CONFIDENTIAL | 14

Next steps ● To confirm those results with other influenza vaccine strains CONFIDENTIAL | 15