recombinant np and np-m2e protein expressed in e.coli ......e.coli np a/jingke/30/95 nm2e wb...
TRANSCRIPT
Recombinant NP and NP-M2e protein expressed
in E.coli elicited robust immunity and broad
protection against influenza A virus in mice
Li Ruan
Wenling Wang Baoying Huang Xiuping Wang Renqing Li Tao Jiang Wenjie Tan
Xiangrong Qi Yingying Gao
National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention (China CDC)
The First WHO Integrated Meeting on Development and Clinical Trials of Influenza Vaccines that
Induce Broadly Protective and Long-Lasting Immune Responses
24-26 January , 2013
HA
NA
HI≥40 Egg-based
Current Seasonal Flu vaccine Challenges
Limited cross-protection efficacy
Limited vaccine availability
Time-consuming (4-6 Months, annually)
Unable to meet the need of control for unpredictable flu
pandemic, even for the mis/poor-matched flu epidemic
Concept of Our Universal Flu Vaccine
Broadly cross-protection efficacy
Large amount vaccine availability
Time-saving production (one for all)
NP
M2e
CTL/ADCC/bNAb
M1
sHA
……
Future Universal Flu vaccine Advantages
Goals: To meet with the control for unpredictable flu
pandemic, even for the mis/poor matched flu epidemic.
Non-major Protective Antigen strategy: Switch our focusing on the vaccine antigen from HA/NA
(major protective antigen in nature infection ) to M2e/NP/sHA et al (non-major protective antigen
in nature infection ), enhancing the immunogenicity of those antigens with adjuvant.
★ The potent of E.coli expressed NP/NP-M2e protein used as universal influenza vaccine?
★ The broad-protection efficacies induced by the E.coli expressed NP/NP-M2e protein?
E.coLi
NP
NM2e
WB
NP Mice serum M2e McAb SDS-PAGE
NP NM2e
2. supernatant
Both recombinant protein of NP and NM2e could be efficiently expressed in E.coli in soluble form;
The untagged soluble proteins of NP and NM2e could be successfully purified and immunogenic.
Characterization of NP and NM2e purified from E. coli
1. whole cell lysate 3. precipitate
W B
NP NM2e NM2e
NP
NP M2e
A/Jingke/30/95
(H3N2)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100PBS
10ugNP
30ugNP
90ugNP
RVJ1175NP
10ug NM2e
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
PBS
30ugNP
90ugNP
RVJ1175NP
10ugNP
10ugNM2e
Days Post Challenge
Surv
ival P
erc
ent (%
)
**
*
36% (4/11)
78% (7/9)
91% (10/11)
58% (7/12)
73% (8/11) *
*
Comparable cross-protective efficacies
against challenge of PR8 were induced by
E.coli expressed NP/NM2e with that of the
RVJ1175NP-immunized mice;
NM2e elicited better protective efficacy than
NP unadjuvanted or adjuvanted with Al (OH)3 ,
CpG, or Al (OH)3 plus CpG
As the adjuvant of Al(OH)3 is a safety and
successful adjuvant for human,
NM2e+Al(OH)3 was used for future study
Protective Efficacy induced by the E.coli expressed NP/NM2e protein was
comparable with that of the Vaccinia based NP vaccine RVJ1175NP in mice
10 MLD50 PR8 challenge
20 MLD50 PR8 challenge
*P≦0.05; ** P≦0.01
0.125 0.25 0.5 1 2 4 8 16 320
20
40
60
80
100
120
Vaccine Doses-log2(ug)
Seru
m H
I Tite
r (G
MT)
3 10 30 100 300 1000 3000
0
20
40
60
80
100 NS-A1
H3-15ug-A1
H1-0.15ug -A1
H1-0.5ug-A1
H1-1.5ug-A1
H1-5ug-A1
H1-15ug-A1
H1-1.5ug-PR8
Virus Challenge Dose(LD50)
Surv
ival P
erc
ent(
%)
Vaccine Evaluation system based on seasonal Flu vaccine
ED50a : 1.5µg HA of seasonal influenza split vaccine could induce HI=40 after one dose in mice.
ED50b: 1.5µg HA could effectively protect mice against 1000LD50 of well-matched hom-influenza virus,
but hardly to protect mice against 3LD50 of poor-matched hom- or het- influenza virus .
HID50=102TCID50(by droplet), MLD50=103-104TCID50; (Fields Virology, 5ed, 2006)
Virus titer in 1MLD50 is about 10-100 times higher than that in the human natural infection by droplet
Based on seasonal Flu vaccine, a efficacy evaluation system for NP/M2e-based vaccine in mice was suggested , that is, immunized with 1.5µg HA of seasonal Flu vaccine, challenged by the well/poor-matched homologous and heterogonous subtype of Flu virus, and the virus challenge doses could be ranged from 3 LD50 to 1000LD50.
1.5ug HA ~40 HI
A1-P5-E1-H1N1(well-matched)
PR8-H1N1(poor-matched)
A1-P5-E1-H3N2(poor-matched, Het)
Humoral immune response
Cellular immune response
Virus Challenge study
Weight Change/Survival rate NP/M2e(IgG/IgG1/IgG2a)
ELISPOT
NP147-155/NP55-69/M2e
ELISA Challenge
Well-matched homologous (H1N1): A1-P5-E1,GD-P40-E1)
Poor-matched homologous (H1N1: A/PR/8/34)
Mis-matched subtype (H1N2: A3JR-P5-E1)
H1N1:
1.5ugHA
(HI=1:40)
NM2e+Al
2.5ug-5ug-10ug
(Humoral/Cellular)
Comparison Study between the E.coli expressed NM2e
protein with the Seasonal influenza vaccine in mice
Ori-Influenza virus Mice adaptation virus LD50(/50ul)
A/Brisbane/59/2007(H1N1)-like virus A1-P5-E1 3.2×103
A/Guangdong/51/2008(H1N1) GD-P40-E1 1.8×103
A/PR/8/34(H1N1) PR8 2.1×104
A/Jingke/30/95(H3N2) A3JR-P5-E1 1.7×104
Influenza virus adaptation in mice
Vaccine Dosage
(ug)
A1-P5-E1(LD50)
well-matched H1N1 GD-P40-E1(LD50)
well-matched H1N1 PR8(LD50)
poor-matched H1N1 A3JR-P5-E1(LD50)
mis-matched H3N2
10 30 100 30 300 3000 5 10 20 3 10 30
NM2e+Al
2.5 * * * * * * * * * * * *
5.0 * * * * * * * * * * * *
10 * * * * * * * * * * * *
NM2e 10 * * *
* * * * * * * * *
H1N1 1.5 * * *
* * * * * * * * *
Al(OH)3 0 * * *
* * * * * * * * *
NM2e+Al(OH)3
Immunization Program and Groups
Al
10ug
NM
2e
2.5u
gNM
2e+A
l
5ugN
M2e
+Al
10ug
NM
2e+A
l
1.5u
gH1N
1
0
2
4
6
8NP-IgG
NP
-Spec
ific
IgG
Tit
er(l
g)
*
Al
10ug
NM
2e
2.5u
gNM
2e+A
l
5ugN
M2e
+Al
10ug
NM
2e+A
l
1.5u
gH1N
1
0
2
4
6 M2e-IgG
M2e-
Spec
ific
IgG
Tit
er(l
g)
*
*
A
B
Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1
0
20
40
60
80
100NP147-155 CTL
SF
C/m
illio
n c
ells
A
Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1
0
50
100
150
200
250
NP55-69 Th
SF
C/m
illio
n c
ells
Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1 Al
10ug
NM2e
2.5u
gNM2e
+Al
5ugN
M2e
+Al
10ug
NM2e
+Al
1.5u
gH1N
1
0
50
100
150
200
250M2e
SF
C/m
illio
n c
ells
IFN-γ IL-4 IL-10
B
C
*
*
*
Immune responses induced by NM2e+Al(OH)3 vaccine in mice
10ug of NM2e+Al(OH)3 induced the strongest
immune responses in mice
Humoral Immune responses Cellular Immune responses
*P≦0.05
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+A
10ugNM2e+Al
10ugNM2e
Al-10LD50
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al0
10ugNM2e
Al
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
Al-100LD50
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+A
10ugNM2e+Al
Al-30LD50
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
en
t (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al
Al
5ugNM2e+Al
1.5ugH1N1
Days Post ChallengeS
urv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al
Al
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+A
10ugNM2e+Al
10ugNM2e
Al-10LD50
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al0
10ugNM2e
Al
5ugNM2e+Al
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
Al
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+A
10ugNM2e+Al
Al-10LD50
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al0
10ugNM2e
Al
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
1 3 5 7 9 11 13 15 17 19 210
20
40
60
80
100
2.5ugNM2e+Al
10ugNM2e+Al
Al
5ugNM2e+Al
1.5ugH1N1
Days Post Challenge
Surv
ival P
erc
ent (%
)
Protection against lethal challenge of well/poor-matched homologous subtype of
influenza virus strains (Survival ration)
A1-P5-E1 well-matched H1N1
10LD50 30LD50 100LD50
30LD50 300LD50 3000LD50
PR8 poor-matched H1N1
5LD50 10LD50 20LD50
3LD50 10LD50 30LD50
***
**
*** ***
**
***
** *
*** ***
*P≦0.05; ** P≦0.01; P≦0.001
*** ***
** **
*** **
** ***
** *
** ** A3JR-P5-E1
mis-matched H1N2
GD-P40-E1 well-matched H1N1
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
1.5ugH1N1
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
Al
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
1.5ugH1N1
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
Al
Days post ChallengeW
eig
ht C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100 2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
Al
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
10ugNM2e
Days post Challenge
Weig
ht C
hange P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100 2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
Al
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
1 3 5 7 9 11 13 15 17 19 2150
60
70
80
90
100
Al
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
1.5ugH1N1
Days post Challenge
We
ight C
hang
e P
erc
ents
(%)
10LD50 30LD50 100LD50
30LD50 300LD50 3000LD50
5LD50 10LD50 20LD50
3LD50 10LD50 30LD50
Protection against lethal challenge of well/poor-matched homologous subtype of
influenza virus strains (Weight Change)
A1-P5-E1 well-matched H1N1
GD-P40-E1 well-matched H1N1
PR8 poor-matched H1N1
A3JR-P5-E1 mis-matched H1N2
10 L
D50
30 L
D50
100
LD50
30 L
D50
300
LD50
3000
LD50
5 LD
50
10 L
D50
20 L
D50
3 LD
50
10 L
D50
30 L
D50
0
20
40
60
80
100
Al
10ugNM2e
2.5ugNM2e+Al
5ugNM2e+Al
10ugNM2e+Al
1.5ugH1N1
Surv
ival P
erc
ent(
%)
A1-P5-E1 well-matched H1N1
GD-P40-E1 well-matched H1N1
PR8 poor-matched H1N1
A3JR-P5E1 mis-matched HN2
E.coli expressed NM2e fusion protein of influenza A virus adjuvants of
Al(OH)3 could Induce broad-spectrum protection in Mice
NM2e-based vaccine elicited broader cross protection than HA-based vaccine in mice,
especially against the poor-matched or mis-matched heterogonous influenza virus
Protective efficacy of NM2e protein is related
with humoral and cellular response in mice
Immune-protection correlation and immune-lasting analysis
0 20 40 60 80 1000
2
4
6
8 NP
M2e
Survival Percent(%)
Sp
ecific
Ig
G T
iter
(lg
)
0 20 40 60 80 1000
40
80
120
160
200
240
280
320
360
400NP147-155 CTL
NP55-69 Th
M2e
Survival Percent(%)
IFN
-r S
FC
/10
6 S
NM
C
r=0.59, P<0.01, R2=0.49
r=0.99, P<0.01, R2=0.99
r=0.77, P<0.01, R2=0.59
r=0.59, P<0.05, R2=0.35
r=0.91, P<0.01, R2=0.81
4 8 12 16 20 24 28 32 36 40103
104
105
106
107
One Dose
Two-Dose(0-14)
Two-Dose(0-35)
Two Dose(0-56)
Three Dose(0-14-28)
Weeks Post Immunization
NP
specific
antibo
dy titer(
lg)
4 8 12 16 20 24 28 32 36 40100
101
102
103
104
105
One DoseTwo-Dose(0-14)
Two-Dose(0-35)
Two Dose(0-56)
Three Dose(0-14-28)
Weeks Post Immunization
M2e s
pecific
antibo
dy titer(
lg)
NM2e elicited strong and long-term humoral
immune response in mice
Both recombinant protein of NP and NM2e could be efficiently expressed in E.coli
in soluble form.
Comparable cross-protective efficacies were induced by E.coli-base NP/NM2e
protein with that of the vaccinia virus-based NP vaccine in mice.
NM2e-based vaccine elicited broader cross protection than HA-based vaccine in
mice.
Recombinant NP and NP-M2e protein expressed in E.coli elicited robust immunity
and broad protection against influenza A virus in mice, and can be used as a
candidate of universal flu vaccine
Summary and Conclusion
Future work
M2e-ADCC
sHA-NAb
NP-CTL
E.coLi
Acknowledgments High Technology Research and Development Program of China (863-2006AA02A203)
National Institute for Viral Disease Control and Prevention ,China CDC
Biotech Center for Viral Disease Emergency, NIVDC, China CDC
Sinovac Bio-tech limited company