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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
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
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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?
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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)
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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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MLE-PT-RFA-15002-Ms - HI against A/New Caledonia/20/99 (H1N1) results - Post-2 IM (D49)
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
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 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)
MLE-PT-RFA-15002-Ms - HI against A/New Caledonia/20/99 (H1N1) results - 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
A/New Caledonia/20/99 (H1N1)
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)
MLE-PT-RFA-15002-Ms - HI against A/New Caledonia/20/99 (H1N1) results - 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