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

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C1 rHA-TMD from Dyadic Immunogenicity study in mice Test MLE-PT-RFA-15002-Ms Mise 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

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CONFIDENTIAL

C1 rHA-TMD from Dyadic Immunogenicity study in mice Test MLE-PT-RFA-15002-Ms

Mise à jour du 14 sept 2015

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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?

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CONFIDENTIAL

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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

(IU/mL –BRD)

Purity

Recombinant HA from the A/H1N1/New Caledonia/20/99 strain produced in Myceliophthora thermophila (C1) TMB (Dyadic, Engen) 1869 BCA (sp): 791 µg/mL µBradford (sp): 627 µg/mL SRID: 129 µg/mL Titre UHA: 1280/50µL 12

80%

Recombinant HA from the A/H1N1/New Caledonia/20/99 strain produced in baculovirus (Protein Sciences) 1367-057 BCA (Protein Sciences): 686 µg/mL µBradford (sp): 391 µg/mL SRID: 200 µg/mL Titre UHA: 32000/50µL <10

>90%

C1 mock1 TMD-rHA (Dyadic, Engen): AEX fractions 4-10 that eluted before the fractions where HA normally elutes off the column 1860 BCA (sp): 970 µg/mL µBradford (sp): 970 µg/mL SRID: ND Titre UHA: 16 79 N/A C1 mock2 TMD-rHA (Dyadic, Engen): AEX fractions 11-15 where the HA normally elutes off (same salt concentrations that HA normally elutes off) 1860 The most representative C1 mock BCA (sp):1276 µg/mL µBradford (sp): 1650 µg/mL SRID: ND Titre UHA: 32/50µL 2452 N/A

CONFIDENTIAL

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Baculovirus rHA PS lot 1367-057 1 2 C1 rHA TMD 1869 Native PAGE TEM Negative staining 1 2 SDS-PAGE 1 2 1 2 N-glycosylation profile of HA by mass spectrometry

Similar oligomeric state between both rHAs

Evaluation of C1 rHA-TMD from Dyadic Biochemical characterization of the two rHAs

Test MLE-PT-RFA-15002-Ms

HA

CONFIDENTIAL

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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

100µl (50µl /thigh): total 88 mice

  • Products
  • 3-Fold Escalating doses of Protein Sciences-rHA : 1 - 3.33 - 10 -

30 µg (8 mice/group=32 mice)

  • 3-Fold Escalating doses of C1-rHA: 1 - 3.33 - 10 - 30 µg (8

mice/group=32 mice)

  • Negative control groups
  • PBS (5 mice)
  • C1 mock preparation 1: 30 µg (5 mice)
  • C1 mock preparation 2 : 30 µg (2 mice)
  • Products
  • “Spiked” group: 10 µg of Protein Sciences- rHA diluted in 30 µg of

C1 mock preparation 1 (8 mice)

  • “Spiked” group: 10 µg of Protein Sciences- rHA diluted in 30 µg of

C1 mock preparation 2 (4 mice)

D28

IM immunizations

D0 D49

  • HI on D27 and D49 serum samples

 

Statistical analysis

  • First: Dose-effect establishment using a model
  • f covariance (dose factor after log10

transformation as continue variable). Comparison by analysis of variances of HI responses induced by the two rHAs post-1 and post-2 injections (IM)

  • Second: evaluation of the adjuvant-effect by

both C1 mock preparations on the Protein Sciences-rHA

19/05/2015 (W21) 15/06/2015 16/06/2015 (W25) 07/07/2015 (W28)

D27

CONFIDENTIAL

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

CONFIDENTIAL

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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

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

CONFIDENTIAL

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µg of A/New Caledonia/20/99 rHA (µBradford dosage)

1 3.33 10 30 1 3.33 10 30 10 10

HI titer against A/New Caledonia/20/99 (H1N1) virus (cRBC)

10 100 1000 11 13 40 123 10 6 10 18 52 40 5 5 5

A/New Caledonia/20/99 (H1N1)

rHA-TMD Myceliophtora Thermophila (Dyadic - Engen) rHA Baculovirus (Protein Sciences) Negative Controls

PBS

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C1 mock1 spiked C1 mock2 spiked C1 mock1 C1 mock2

HI response after 1 IM immunization in mice: Statistical analysis was not possible due to too much non responders Although:

  • Clear evidence of the superiority of

C1-rHA TMD over baculo-rHA (number of responders and mean titers) Not statistically analyzed C1-rHA TMD > Baculo-rHA

30µg 30µg

Immunogenicity evaluation of C1 rHA-TMD Results: A/NC/20/99 HI specific response post-1 IM (D27)

1µg 3.33µg 10µg 30µg C1 50% (4/8) 57.1% (4/7) 100% (8/8) 100% (8/8) Baculo 62.5% (5/8) 12% (1/8) 50% (4/8) 75% ( 6/8 ) Dose of rHA Expression system

Percent of responders

  • CONFIDENTIAL
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µg of A/New Caledonia/20/99 rHA (µBradford dosage)

1 3.33 10 30 1 3.33 10 30 10 10

HI titer against A/New Caledonia/20/99 (H1N1) virus (cRBC)

10 100 1000 108 104 453 830 95 44 123 415 640 381 5 5 5

A/New Caledonia/20/99 (H1N1)

rHA-TMD Myceliophtora Thermophila (Dyadic - Engen) rHA Baculovirus (Protein Sciences) Negative Controls

PBS

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C1 mock1 spiked C1 mock2 spiked C1 mock1 C1 mock2

HI response after 2 IM immunizations in mice:

  • Significant 2-fold difference in favor of

C1-rHA TMD (p-value: 0.006) Significant difference : C1-rHA >baculo-rHA

Immunogenicity evaluation of C1 rHA-TMD Results: A/NC/20/99 HI specific response post-2 IM (D49)

30µg 30µg

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

  • CONFIDENTIAL
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Statistical analysis was not possible due to too much non responders

  • Both C1-mock1 and C1-mock 2

spiked groups (30µg) showed higher responses than those measured with 10 µg of baculo-rHA alone Not statistically analyzed

Evaluation of the matrix effet of C1 rHA-TMD at high dose Results: A/NC/20/99 HI specific response post-1 IM (D27)

HI titer against A/New Caledonia/20/99 (H1N1) virus (cRBC)

10 100 1000 10 52 40 5 5 5

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C1 mock1 spiked C1 mock2 spiked

baculo-rHA (10µg) Negative Controls PBS C1 mock1 (30µg) Spiked groups baculo-rHA (10 µg) in C1 mock2 (30µg) C1 mock1 (30µg) C1 mock2 (30µg)

Mock2 most representative C1 mock

  • CONFIDENTIAL
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HI response after 2 IM immunizations in mice:

  • Significant adjuvant-effet with both

C1-mock preparations (30µg) on the baculo rHA (10µg)

  • with C1-mock 1 (p-value: 0.0005)
  • with C1 mock-2 (p-value: 0.0439)

Significant difference : Adjuvant-effect with both C1-mock preparations

Evaluation of the matrix effet of C1 rHA-TMD at high dose Results: A/NC/20/99 HI specific response post-2 IM (D49)

HI titer against A/New Caledonia/20/99 (H1N1) virus (cRBC)

10 100 1000 123 640 381 5 5 5 baculo-rHA (10µg) Negative Controls PBS

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C1 mock1 (30µg) Spiked groups baculo-rHA (10 µg) in C1 mock2 (30µg) C1 mock1 (30µg) C1 mock2 (30µg)

Mock2 most representative C1 mock

  • CONFIDENTIAL
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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

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CONFIDENTIAL

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Next steps

  • To confirm those results with other influenza vaccine strains

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CONFIDENTIAL