Study of molecular epidemiology and vaccine development for enterovirus 71 in Mongolia

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Janchiv Oyunbileg,3 Chun-Nan Lee1,2,

O.Dulamsuren3, P.Suvd3, Yi-Hsin Yao1, Bin-Ching Ho1,

Z.Sainjargal3, B.Enkhtuya3, Sui-Yuan Chang,1,2 and

1Department of Clinical Laboratory Sciences and Medical Biotechnology,

College of Medicine, National Taiwan University; 2Department of Laboratory

Medicine, National Taiwan University Hospital; Taipei, Taiwan, Republic of

China; 3Public Health Institute of Ministry of Health, Ulaanbaatar, Mongolia.

Introduction • Enterovirus 71 (EV-71)

•

Enterovirus 71 (EV-71)

•Outbreaks occurred worldwide especially in the Asia-Pacific region

•High morbidity and mortality

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Enterovirus 71 (EV-71)

•No commercial antiviral therapy or vaccine

•Rely on public health surveillance and quarantine

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Enterovirus 71 (EV-71)

•Picornaviridae

• (+)ssRNA

•7,500 bases

•Non-enveloped

•Capsid : VP1, VP2, VP3 and VP4

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VP1 protein

•On the surface of virion

•Targeted by host neutralizing antibodies

→ important in viral pathogenesis and virulence

Based on the VP1 gene

•Genogroups : A, B, and C

• Subgenogroups : genogroup B - B1 to B5

genogroup C - C1 to C5 6

VP1 protein • Involved in the recognition of EV71 receptors

•Displays major antigenicity Sivasamugham LA et al., 2006

•Antibodies against VP1 are important for virus neutralization.

Tan CS et al., 2007; Tung et al., 2007; Damian et al., 2006

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Adenovirus

• DNA virus

• Non-enveloped

• Capsid : 12 pentons and 240 hexons

• 36-kb double-stranded linear DNA

• Inverted terminal repeat (ITR) sequences at each end

penton

hexon

viral genome

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Advantages of using recombinant adenovirus

1. Broad host range and low pathogenicity in humans.

2. Replicates efficiently to high titers

3. Helper independent Ad can accommodate up to 7.5 kb of foreign DNA.

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Advantages of using a recombinant adenovirus

4. Proper folding and exact post-translational modifications of human proteins

5. No insertional mutagenesis; remains epichromosomal

6. Infection and expression of genes in replicative and non-replicative cells.

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Aims

• Phylogenetic analysis of EV71 strains isolated in Mongolia from 2008 to 2009

• Generate recombinant adenovirus expressing VP1 protein

• Test its immunogenicity against EV71 in mice

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Phylogenetic analysis of EV71 Strains isolated in Mongolia from 2008 to 2009

• 8 isolates of 2008

• 2 isolates of 2009

• Based on the VP1 gene

• Subgenotype C2

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C2

C3

C5

C1

C4

A

B1

B3

B4

B5

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C2 VP1 200bp

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Generation of Recombinant

Adenovirus Using AdEasy TM

Experimental Design

• EV71 RNA -------> VP1 gene

RT-PCR

Overlapping

PCR

Gene of interest

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VP1 Gene Constructs

Signal peptide 66bp

VP1 891bp/40KD

Fc 678bp/30KD

Linker Linker CT 375bp/ 13.8KD

Signal peptide VP1

Signal peptide VP1 Fc

Linker

Signal peptide VP1 Linker CT

Signal peptide Fc Linker CT

Construct 1 2115bp

Construct 3 1389bp

Construct 2 1695bp

Construct 4 969bp

Construct 5 1181bp

45bp 45bp

Construct MN 969bp

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Generation of a Recombinant Adenovirus Using AdEasy TM

BJ5183

In vivo homologous recombination In BJ5183

pShttle-CMV

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Generation of Recombinant Adenovirus Using AdEasy TM

In vivo homologous recombination In BJ5183

Check step: 1. PCR 2. Pac I : 4.5kb

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Generation of Recombinant Adenovirus Using AdEasy TM

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Cytopathic effect

Cell Control Construct 4 of rAd

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Indirect Immuofluorescence Assay • Anti- Adenovirus Hexon Ab

• 200x

1 SP-VP1-Fc-CT

FITC

BF

2

SP-VP1-Fc

3

SP-VP1-CT

4

SP-VP1

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Indirect Immunofluorescence Assay • Anti- Adenovirus Hexon Ab

• 200x

5 SP-Fc-CT

Mock Cell control

BF

FITC

MN SP-MN VP1

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Recombinant Adenovirus Amplification

• Plaque purification for 3 times

• Large scale amplification

• CsCl gradient ultracentrifugation

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CsCl gradient ultracentrifugation

←

←

1st 35,000rpm 4℃ 2hr

2nd 35,000rpm 4℃ 20hr

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Conclusions

• The VP1 genes of the Mongolian isolates are closely related with the Russian strains of 2009.

• Whether the Mongolian EV71 strains were originated from Russia still needs further studies by including more reference strains of Russia and other countries.

• The recombinant adenoviruses expressed VP1 protein well.

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Experimental Design

Gene of interest TA cloning

Sequncing Restriction enzyme digestion

Ligation with pShttle-CMV

pShttle-CMV

•Check step: 1. Sequencing 2. Restriction enzyme digestion

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M 1 2 3 Lane1: Vector control Induced by IPTG Lane2: pLsys/pRSETA/VP1 without IPTG Lane3: pLsys/pRSETA/VP1 Induced by IPTG

43KD

34KD

VP1 :40KD ←

Express VP1 protein in E. coli system

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Lane 1: pLsys/pRSETA/VP1 without IPTG

Lane 2: pLsys/pRSETA/VP1 Induced by IPTG

Lane 3: pellet after sonication

Lane 4: flow through

Lane 5: wash / binding buffer

Lane 6,7: wash / wash buffer (pH6)

Lane 8,9: wash / wash buffer (pH5.3)

Lane 10~17: Eluent / elution buffer (pH4)

←

1 2 3 4 5 6 7 8 9

SDS-PAGE 10 17 11 12 13 14 15 16

← VP1

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Induced by IPTG (1mM) Protein expression

pRSETA- VP1 plasmid in E. Coli BL21(pLsys)

SDS-PAGE, Western blot

Ni2+ column purification

Express VP1 protein in E. coli system

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Western blot

• Anti- His tag Ab

43KD

34KD

VP1 ←

M 1 2

Lane1: pLsys/pRSETA/VP1 without IPTG Lane2: pLsys/pRSETA/VP1 Induced by IPTG

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Western blot

43KD

34KD

M 1 2 3

Lane1: anti-His Ab

Lane2: Mouse serum

EV71 infection

Lane3: Mouse serum

w/o EV71 infection

VP1 ←

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ELISA

• Antigen : EV71 VP1 protein 1μg/μl 100 μl/well

• 1 °Ab : diluted mouse serum

• 2 °Ab : HRP-goat anti- mouse IgG

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

1/50 1/100 1/200 1/400

Series1

Series3

EV71

infected

mouse

EV71

non-infected

mouse

OD450 P

Mouse Immunization

•6~8-week old female BALB/c mice

•Adenovirus titer: 1x 109 pfu

•Oral administration

• Immunize on day 1 and day 25

• Test : VP1 recombinant adenoviruses

• Control : rADV/control; PBS control

•Blood collection : days 0, 7, 21, 28, 42

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Detect anti-VP1 IgG in mice by ELISA (1:50 dilution of serum)

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A: Immune mice with rADV 1/ SP-

VP1-Fc-CT

B: Immune mice with rADV 2/ SP-

VP1-Fc

C: Immune mice with rADV 3/ SP-

VP1-CT

D: Immune mice with rADV 4/ SP-

VP1

E: Immune mice with rADV 5/ SP-Fc-

CT

F: PBS control

Comparison of immune responses among different groups of mice

(1:50 dilution)

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Detect anti-VP1 IgG in mice by ELISA (1:100 dilution of serum)

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A: Immune mice with rADV 1/ SP-

VP1-Fc-CT

B: Immune mice with rADV 2/ SP-

VP1-Fc

C: Immune mice with rADV 3/ SP-

VP1-CT

D: Immune mice with rADV 4/ SP-

VP1

E: Immune mice with rADV 5/ SP-Fc-

CT

F: PBS control

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Comparison of immune responses among different groups of mice

(1:100 dilution)

Results of Mouse Immunization

• Only rADV 3/ SP-VP1-CT could induce a portion of the immunized mice to produce anti- VP1 IgG.

• No neutralizing antibodies were induced.

– not sufficient viral dosages administered

– harsh environment of digestive tract might have degraded the epitopes of VP1 protein

– recombinant adenoviruses did not produce enough target protein

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