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
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Recombinant NP and NP-M2e protein expressed in E.coli ......E.coLi NP A/Jingke/30/95 NM2e WB SDS-PAGE NP Mice serum M2e McAb NP NM2e 2. supernatant Both recombinant protein of NP and
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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 .
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.