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