C1 rHA-TMD from Dyadic Immunogenicity study in mice Test ... · | 1 CONFIDENTIAL C1 rHA-TMD from...

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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

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


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


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


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

of 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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MLE-PT-RFA-15002-Ms - HI against A/New Caledonia/20/99 (H1N1) results - Post-1 IM (D27)

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

1 3.33 10 30 1 3.33 10 30 10 10

HI

tite

r ag

ain

st A

/New

Cal

edo

nia

/20

/99

(H

1N

1)

vir

us

(cR

BC

)

10

100

1000

1113

40

123

10

6

10

18

5240

5 5 5

A/New Caledonia/20/99 (H1N1)

rHA-TMD

Myceliophtora

Thermophila

(Dyadic - Engen)

rHA Baculovirus

(Protein Sciences)Negative Controls

PBS

5

C1mock1spiked

C1mock2spiked

C1mock1

C1mock2

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 rHAExpression

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

tite

r ag

ainst

A/N

ew C

aled

onia

/20

/99 (

H1N

1)

vir

us

(cR

BC

)

10

100

1000

108 104

453

830

95

44

123

415

640

381

5 5 5


rHA-TMD

Myceliophtora

Thermophila

(Dyadic - Engen)

rHA Baculovirus

(Protein Sciences)Negative Controls

PBS

5

C1mock1spiked

C1mock2spiked

C1mock1

C1mock2

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

tite

r again

st A

/New

Cale

do

nia

/20

/99 (

H1N

1)

vir

us

(cR

BC

)

10

100

1000

10

5240

5 5 5


5

C1mock1spiked

C1mock2spiked

baculo-rHA

(10µg)

Negative Controls

PBSC1 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

tite

r ag

ainst

A/N

ew C

aled

onia

/20

/99 (

H1N

1)

vir

us

(cR

BC

)

10

100

1000

123

640

381

5 5 5

baculo-rHA

(10µg)

Negative Controls

PBS

5

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

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

Next steps

● To confirm those results with other influenza vaccine strains

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CONFIDENTIAL

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