h7n9 influenza and enterovirus 71 (ev71) virus vaccine ... against c4 viruse challenge pbs 2+...
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H7N9 influenza and Enterovirus 71 (EV71) virus vaccine development in Taiwan
The Head of R&DJuine-Ruey Chen Ph.D
國光生物科技(股)公司ADIMMUNE CORPORATION
Major Products (1) JEV vaccine, domestic market share : 100%
(2) Tuberculin PPD, domestic market share : 100%
(3) Tetanus toxoid, domestic market share : 50%
(4) Influenza vaccine, domestic market share : 30%
Founded in 1965 45 years of expertise in Vaccinology
Company location Taichung
Business Focus Vaccines, Diagnostics, & Biological Products
Primary Business Developer / Manufacturer / Distributor
Technology Partner Crucell , Kitasato Institute
The only cGMP manufacturer and company of human vaccine in Taiwan
Overview of the Adimmune
H7N9 raise pandemic concernsand the vaccine preparation
• By 29 May 2013, approximately 2 months after the initial report, the numberof laboratory-confirmed H7N9 infections reached 132, with 37 deaths.
• The case fatality proportion reached approximately 25%, which is a provisionalvalue because many patients remain hospitalized as of 8 May 2013 and thenumber of mild cases remains unknown.
• However, most patients had a history of recent exposure to poultry, generallyat live bird markets, however there is no evidence of sustained onwards virustransmission to other people.
• The concern for a pandemic caused by a newly emerged avian influenza A virushas led to clinical trials with H7N9 candidate vaccines .
• The Minister of health, Taiwan supported two grants for the Taiwan vaccineindustry to develop the H7N9 vaccines.
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
Conclusion: This inactivated subunit influenza A (H7N7) vaccine was safe but
poorly immunogenic in humans
The challenge of development of H7 influenza vaccine
(Sanofi Pasteur Inc)
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
Seed virus:E4 or E5 10 7~8 EID50
10-3 dilution 10-4 dilution 10-5 dilution 10-6 dilution
0.2mL/embryo egg 0.2mL /embryo egg 0.2mL /embryo egg 0.2mL /embryo egg
34±0.5℃
30hr48hr
52hr
5 eggs/group
Allantoic fluid collection
初離心移除卵蛋白8,000g 4℃ 30min
超高速離心收集病毒(Pellet down)
130,000g 4℃ 60min
30hr48hr
52hr
30hr48hr
52hr
30hr48hr
52hr
To estimate the HAU titers
Investigation of the best incubation condition for the H7N9 virus production
Improvement of the H7N9 vaccine efficacy in Adimmune Corp.
Al(OH)3
Squalene
w/o adjuvant
(1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg(3) Human 90µg/ dose 3.0 µg
Inactivated split and whole H7N9 virus vaccine
(1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg(3) Human 90µg/ dose 3.0 µg
(1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg(3) Human 90µg/ dose 3.0 µg
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
The electron micrograph of A/Mallard/Netherlands/12/2000-IBCDC-1(H7N7)
A/Shanghai/2/2013 IDCDC-RG32A (H7N9) inactivated split or whole viruses
How to prepare an effective influenza vaccine
with glycoprotein HA
Chen JR, Ma C, Wong CH. Trends Bio technol. 2011 Sep;29(9):426-34.
Purification of HA protein of H7N9 with Ni-NTA column
30 60 90 150 300 500 IM(mM)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Adimmune
Digestion of H7N9 extracellular domain of HA with N-glycosidase
100
70
55
40
130
33
25
15
Endo H cleave only high mannose structures (n = 2-150, x =
(Man)1-2, y = H)
M 2 5 10 20 30 60 min
Endo H
100
70
55
40
130
170
PNGase F hydrolyzes nearly all types of N-glycan chains from
glycopeptides/proteins. [x = H or sugar(s)]
M 0 1 3 5 7 9 20 30 40 60 min
PNGase F
Purification and characterization of the H7N9 extracellular domain of HA
ADIM-rHA
ADIM-Whole
Quantification of the H7N9 vaccine bulk for clinical trial
Split H7N9 HA Standard curve
y = 9.7017x + 0.2575
R2 = 0.9911
0.0000
0.1000
0.2000
0.3000
0.4000
0.5000
0.6000
0.7000
0.8000
0.9000
1.0000
0.0000 0.0200 0.0400 0.0600 0.0800
HA Conc (ug/ml)
OD45
0 Standard(RD SplitH7N9)
線性 (Standard(RDSplit H7N9))
336.06
Sample nameSample HAConc(μg/ml)
Sample totalprotein(μg/ml)
FAS13001 Split H7N9 312.84 246.8FAS13002 Split H7N9 291.2FAS13003 Split H7N9 318.55 284.4
ELISA SRIDMethods
322.5±12.1 274.1±23.9
Immunogenicity of H7N9 vaccine in mice
A
B
C
14 28 35 50
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
Inactivated H7N7 split and whole virus antigens combined with different adjuvants as a vaccine in mice (1)
(Split
) 0.5
ug w/o 3
(Split
) 0.5
ug +Al(O
H)
(Split
) 0.5
ug +Addavax 3
(whole
) 0.5
ug +Al(O
H)
(whole
) 0.5
ug +Addavax
(Split
) 1.5
ug w/o
(Split
) 1.5
ug +Al(O
H)3
(Split
) 1.5
ug +Addavax
(whole
) 1.5
ug +Al(O
H)3
(whole
) 1.5
ug +Addavax
(Split
) 3ug w
/o
(Split
) 3ug +
Al(OH)3
(Split
) 3ug +
Addavax
(whole
) 3ug +
Al(OH)3
(whole
) 3ug +
Addavax0
200
400
600
800
1000
1200
Ser
um
HI t
iters
(G
MT
)
0.5ug 1.5ug 3ug
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
(Split) 0.5ug
w/o 3
(Split) 0.5ug
+Al(O
H)
(Split) 0.5ug
+Add
avax
(who
le) 0
.5ug
w/o 3
(who
le) 0
.5ug
+Al(O
H)
(who
le) 0
.5ug
+Add
avax
Non
- immun
ized
con
trol
0
500
1000
1500
2000
2500
3000
Ne
utr
ali
za
tio
n t
ite
rs
(G
MT
)
(Split) 0.5ug
w/o 3
(Split) 0.5ug
Al(O
H)
(Split) 0.5ug
Add
aVax
(Who
le) 0
.5ug
w/o
(Who
le) 0
.5ug
Al(O
H)3
(Who
le) 0
.5ug
Add
aVax
Non
-immun
ized
con
trol
0
500
1000
1500
2000
2500
(Split) 0.5ug
w/o 3
(Split) 0.5ug
+Al(O
H)
(Split) 0.5ug
+Add
avax
(who
le) 0
.5ug
w/o 3
(who
le) 0
.5ug
+Al(O
H)
(who
le) 0
.5ug
+Add
avax
Non
- immun
ized
con
trol
0
500
1000
1500
Vaccine: H7N9 Shanghai-2Virus: H7N9 Shanghai-2
Vaccine: H7N9 Shanghai-2Virus: H7N9 Anhui-1
Vaccine: H7N9 Shanghai-2Virus: H7N7
H7N9 (Shanghai-2) vaccine not only elicited neutralizing antibodies against same strain of virus but also conferred cross-protection against
H7N9 (Anhui-1) and H7N7 viruses
HAI≧40
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
0 1 2 3 4 5 6 7 8 9 10111213140
102030405060708090
100110
AddaVax
0.5 g H7N9-S
0.5 g H7N9-S-Al(OH)3
0.5 g H7N9-S-AddaVax
0.1 g H7N9-S-AddaVax
Su
rvia
l R
ate
(%
)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
10
20
30
40
50
60
70
80
90
100
110
days after infection
Bo
dy w
eig
ht
(%)
H7N9 Virus Challenge studies in vaccine immunized mice
0 1 2 3 4 5 6 7 8 9 10111213140
102030405060708090
100110
AddaVax
0.5 g H7N9-S
0.5 g H7N9-S-Al(OH)3
0.5 g H7N9-S-AddaVax
0.1 g H7N9-S-AddaVax
Su
rvia
l R
ate
(%
)
Positive Results Presented from H7N9 vaccine with adjuvants in Clinical Trial
Summary
國光生物科技(股)公司ADIMMUNE CORPORATION
國光生物科技(股)公司ADIMMUNE CORPORATION
1. The H7N9 inactivated virus vaccine is sufficient to confer cross-neutralizing immunity against H7N9 and H7N7 viruses
2. The Squalene-based H7N9 vaccine may be the best formulation for the further vaccine preparation
3. Adimmune alum-based H7N9 vaccine results showed that the seroprotection rate were met with the criteria for Phase II clinical and will conduct the phase III study in 2015.
The Development of EV71 Inactivated Virus Vaccine with
Single Use Bioreactor
國光生物科技(股)公司ADIMMUNE CORPORATION
Juine-Ruey Chen Ph.D陳俊叡
(A) Host:Vero cells(B) Virus:Validated virus seed from NHRI.(C) Adimmune has successfully established the EV71 vaccine production with 10L and 50L bioreactors.(D) Adimmune was supported by Taiwan government to speed up the progress of EV71 vaccine
development, 200L and 1000L production scale will be established in 2014.
The scale up the production scale for preparation of EV71 vaccine in phase II clinical trials
Production of EV71 vaccines with 200L single use bioreactor
R&D Department ManagerJuine-Ruey Chen Ph.D
陳俊叡
國光生物科技(股)公司ADIMMUNE CORPORATION
Adimmune
Production of EV71 vaccine with 200L Bioreactor has
been successfully established by Adimmune R&D in NHRI
Adimmune
Production of EV71 vaccine with 200L Bioreactor has been
successfully established by Adimmune R&D in NHRI
Virus challenge to test if the EV71 vaccine provide sufficient protection in mice
國光生物科技(股)公司ADIMMUNE CORPORATION
ELISpot
Cross-protection of Adimmune EV71(B4; E59) vaccine
EV71(C2) challengeEV71(B4) vaccination
PBS group
Vaccine group:
-- 2 g EV71+Al(OH)3
-- 6 g EV71+Al(OH)3
-- 2 g EV71+ AddaVAX
Current status (D35):
All die All survival
C2 (Asia strain)
Adimmune-B4
(Bioreactor)
Mice immunized vaccine Virus challenge
B4 (Taiwan)
C4 (China strain)
Adimmune EV71 (B4) vaccine conferred cross-protective
immunity against C2 and C4 viruses challenge
(A)
(B)
0 2 4 6 8 10 12 14 160
102030405060708090
100110
2 g B4+Al(OH)3
6 g B4+Al(OH)3
PBS
Vaccine: B4
Virus: C2
Su
rv
ial R
ate
(%
)
0 2 4 6 8 10 12 14 16-200
-100
0
100
200
2ug+Al(OH)3
6ug+Al(OH)3
PBS
days after infection
Bo
dy
We
igh
t (%
)Adimmune EV71 (B4) vaccine conferred cross-protective
immunity against C2 virus challengeAdimmune EV71 (B4) vaccine conferred cross-protective
immunity against C2 viruse challenge
0 1 2 3 4 5 6 7 8 9 1011121314150
102030405060708090
100110
PBS2ug+Al(OH)36ug+Al(OH)3
Vaccine: B4
Virus: C4
days post-infection
Su
rviv
al ra
te (
%)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15-50
0
50
100
150
PBS
2ug+Al(OH)36ug+Al(OH)3
days post-infection
Bo
dy
we
igh
t (%
)
6μg vaccine
2μg vaccine
PBS
Adimmune EV71 (B4) vaccine conferred cross-protective
immunity against C4 virus challenge
Day 6 post infection
(A)
(B)
Adimmune EV71 (B4) vaccine conferred cross-protective
immunity against C4 viruse challenge
PBS 2+ AddaVAX 2+AlPO4 6+AlPO4
Mock
C2
C4
The features of pathological changes in muscles of EV71 infected
hSCARB2-Tg mice.
In situ EV71 distribution in the brainstem of hSCARB2-Tg mice
The GMP pilot production plan of EV71 vaccine
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