NOVEL STRATEGIES FOR IMMUNIZATION AGAINST FOOT-AND-MOUTH DISEASE

Dr Srinivas GarlapatiDVM PhD

INDIAN VETERINARY RESEARCH INSTITUTEBengaluru

Economic and Food Security Impact of

Foot-and Mouth Disease 

1. High contagious nature2. Dramatic effect on productivity

   i.  Milk Yield ii. Draught Power3. Antigenic Variability of the pathogen

1. TESTS FOR DIAGNOSIS AND SURVEILANCE, 2. VACCINOLOGY

The 1996 FAO Expert committee recommendations on Improved & Cost-effective

approaches for control of FMD

FMDVLIFE CYCLE Complexity & Evolution call for a new generation of Anti-FMD vaccines (Domingo, 2002).

FMDV Control 1. Conventional Vaccines: Risks, High Costs, Carrier Status2. Non-conventional strategies: I Synthetic peptidesII Rec. Protein based vaccines: Subunit vaccines, DNA based vaccines3. Expression Systems:

BacterialInsect & Yeast cells

Plant and Mammalian expression systems

Live Viral Vectors(DNA & RNA viruses) Insertion of Antigen genes: Deletion of Non-essential and Essential genes Advantages:Safety – No Exposure to the Disease Agent itselfSafety by vector – Attenuated/

Non-ReplicativeVaccinia expressing P1, VP1 (Berinstein, 2003, Sanz para, 1999)

Promote Strong Immune Responses: 1.      Antigen Exp. in Natural host : Post-translational mod.’s – Broad & efficacious immune responses.2.      Infection with tissue destruction and Inflammation – Activate immune responses.3.      Poor Immunogens manipulated to enhance Immunogenicity. 4.      No Adjuvant5.      Cheap – Multivalent

Poxvirus vectors 1. Safety: Replicate in the Cytoplasm2. Recombinants:

Homologous DNA recombination3. Insertion of foreign DNA (25,000 bp)4. Potential Vaccine candidates used in Human and Veterinary medicine Eg. Racoonpox, Swinepox, Capripox,

Success Stories in Veterinary Medicine 1. RABORAL V-RG: Vaccinia exp. Rabies Glycoprotein G 2. Eradication of Rabies in the wild

TROVAC of Fowlpox3. Licensed by USDA: TROVAC-NDV & TROVAC-AI-H5.4. ALVAC expressing HA and F antigens of CDV in Dogs5.Rabies G has been licensed for use in Cats

FMDV and Sheep Sheep Incriminated as carriersNo contact transmissions but are Reservoirs:Potential source of antigenically altered virus variants Mixed farming: Cattle with Sheep For Complete Eradication, Vaccination of sheep is also important

 Live attenuated sheeppox vaccine (SPV-RF) in IndiaVaccine induced disease & Restrictions on Use Need for more AttenuationDisruption of Non-essential gene More attenuation – more safetyNo Marker :Need for a marker vaccine

Sheeppox is major concern in Sheep

Insert FMDV genes into SPV vaccine strainGFP Marker Dual Vaccine (Sheeppox and FMD)

Present study Aims at

OBJECTIVES 1.  Construction of transfer vector with the C7L gene of SPV-RF

2.  Insertion of marker gene, GFP 3.  Cloning of P1-2A gene into the transfer vector and subsequently into the sheeppox virus4.  Studying the immune response of the sheeppox virus vectored FMD vaccine.

Poxvirus Transfer Vectors in General

FL FR

Promoter

Multiple Cloning Sites

Marker (selection)

Construction of SPV Transfer Vector

HR-C7L-L

Gene of interest

GFP HR-C7L-R

FMDV-IRESC7L-Promoter

SPV Genome Organization

Amplification of Sheeppox virus C7L gene

Cloning of C7L geneColony lysis R.E. Digestion

2,4 - Recombinants 1,2,3 - Recombinants

Sequencing

Fig 6 Restriction map of HR (C7L) gene

Sac I AleI SacII BstXI EagI BsrB1XbaI SpeI

BamHI BamHI

XmaI SmaI PstI EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI

SacI XbaI XmaI SmaI PstI EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI

BamHIBamHI

3. Religation (T4 DNA Ligase

1. Sac I & XbaI Digest 2. T4 filling

BamHIBamHI

XmaI SmaI PstI EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI

4. SmaI & KpnI Digest 5. T4 filling

BamHIBamHIBamHI

XmaI Sma I KpnI

6. Religation (T4 DNA Ligase)

Xma I

BamHIBamHI

Strategy for Neutralization of RE sites in the vector

Xma I

BamHIBamHI

SpeI

SpeI

SpeI

SpeI

SpeI

SpeI

Cloning of MCS-EGFP

M – Marker

1 – EGFP

2 - EGFP

3 – Negative control

M 1 2 3

750 bp

Cloning of UTR-GFP in pHGColony Lysis RE Digestion

1.2 kbp

4.5 kbp

1000 bp750 bp

250 bp

500 bp

2000 bp

Colony lysis

M 1 2 3 4

Colony PCR

RE Digestion

1000 bp

500 bp

1100 bp

1500 bp

Xma I

BamHIBamHI

SpeI

Hpa I

MCS GFP

HR

3/-A

3/-A

PCR

7. Hpa I Dig. 8. T4 polishing

HR-L HR-R

9. T4 DNA ligation

MCS

MCS

MCS GFP

GFP

HR-R HR-L

250

50075010002000

1200 bp

Colony Lysis RE Digestion

Cloning of UTR-GFP in pHG

HR-C7L-L

EGFP

EGFP HR-C7L-R

FMDV-IRESC7L-Promoter

GFP expression studies

Normal LT cells Mock transfected Negative control

pCX1-EGFP

pHUGG

pCUG

CMV promoter

SPV-HR(C7L)promoter

CMV promoter with FMDV IRES

10 x 40 x

Cloning of P1-2A in Transfer vector

Colony Lysis

1-6 Recombinants7 – Non-recombinant

4.5kbp3.0kbp2.5kbp2.0kbp

2.8 kbp

HR-C7L-L

P1-2A

GFP

FMDV-IRESC7L-Promoter

HR-C7L-R

Use of Entire P1-2A50 100 150 200 250 300 350 400 450 500 550 600 650 700

Alpha, Regions - Garnier-RobsonA

Alpha, Regions - Chou-FasmanA

Beta, Regions - Garnier-RobsonB

Beta, Regions - Chou-FasmanB

Turn, Regions - Garnier-RobsonT

Turn, Regions - Chou-FasmanT

Coil, Regions - Garnier-RobsonC

Hydrophilicity Plot - Kyte-Doolittle0

4.5

-4.5

Alpha, Amphipathic Regions - Eisenberg*

Beta, Amphipathic Regions - Eisenberg*

Flexible Regions - Karplus-SchulzF

Antigenic Index - Jameson-Wolf0

1.7

-1.7

Surface Probability Plot - Emini1

6

M

pUP pHUPG

C

72 kDa

95 kDa84 kDa

Recombinant virus(rSPV) Generation

Tranfection of LT monolayers:

•pHUPG plasmid •SPV DNA •Escort-III Transfectin

Screening for Recombinants:

•10-1 to 10-10 Virus dilutions infected to LT cells• All showed positive by PCR •The virus form the final 10-10 used for cloning

Limiting dilution method used for picking recombinants:

• Cell-Virus Lysate distributed to 24 well LT-cells• Virus from each well screened for recombinants•PCR for recombinants using GFP and VP1 primers•Simultaneous screening for non-recombinants using HRprimers

HR-L

HR-R

VP1-L

2A-R

GFP-L

GFP-L

HR-L

HR-R

660 bp 750 bp

4.3 kbp

720 bp

PCR for 660bp VP1 gene

PCR for 720 bp HR gene

1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 20

VP1 - - - - - - - + - - + + + + + - + + + -

GFP - - - - - + - + - - - + + - - - - + - -

HR - - + + - - - - + - + - + + - + + - - +

WB + + + -

• 1-20 : well no’s from 24 well plate• VP1- PCR for 660 bp VP1• GFP – PCR for 750 bp GFP• HR – PCR for HR gene• WB – Expression of P1 by Western Blotting

Summary of screening for recombinants and non-recombinants

2nd round of screening for recombinants & non-recombinants

GFP 750 bp

VP1 660 bp

HR 720 bp

Western Blotting for P1-2A Expression

72 kDa95 kDa

84 kDa

1 2 3 4 5 6 7 8 9 10 11 12

VP1 + + + + + + + + + + + +

GFP + + + + + + + + + + + +

HR - - - - - - - - - - - -

WB + + + + + + + + + + + +

Summary of screening for recombinants and non-recombinants after 2nd round

Morphology of rSPV Infective Foci

Wt SPV-RF rSPV

CPE in Recombinant and Non-recombinant SPV

Wt SPV-RF rSPV-RF

Titres 10 log 7.2 10 log 6.8

Immunological Evaluation

Heterologous Prime-Boosting regimen:• Overcome vector Immunity• Different modes of Presentation• Different routes of Inoculation

Live viral vectors are important components of Prime-Boosting regimens:

New strategies for second generation vaccines based on cellular immunity.

Live viral vectors are important??

Group No of Sheep

Vaccine (prime)0 Day

Vaccine(Boost )28 Day

1 (Negative control)

5 PBS --

2 (conventional FMD control)

4 Conventional FMD vaccine

--

3 (conventional

sheep pox vaccine)

2 SPV-RF --

4 rSPV-FMD

5 rSPV-FMD100 TCID50

5protein + rSPV

prime boost

5 FMDV P1 protein(0.6 mg)

+rSPV-FMD

100 TCID50

--

6DNA prime boost

5 DNA vaccine (pUP)0.4 mg

rSPV-FMD100 TCID50

7DNA vaccine

5 DNA vaccine (pUP)0.4 mg

DNA vaccine (pUP)0.4 mg

8Protein vaccine

5 P1 protein(600 μg)

P1 protein(600 μg)

Immunization Schedule

Morphology of rSPV Infective Foci

Wt SPV-RF rSPV

Viral Neutralization Titers

IFN-γ ELISPOT

Long Term Immune Responses

Conclusions1. The C7L gene of Sheeppox virus has been

PCR amplified, Cloned and Sequenced.

2. The C7L promoter has been found to be a strong Early promoter useful for CMI response.

3. The Transfer vector was constructed.

4. Activity of C7L promoter in the Transfer vector has been confirmed by GFP expression

5. Utility of the transfer vector to carry foreign genes has been confirmed by inserting and expressing FMDV P1-2A

6. Cloning of the rSPV has been done without any dominant selection markers.

7.Screening of Recombinant SPV with P1-2A polyprotein gene has been accomplished.

8. Expression of P1-2A in the rSPV has been confirmed.

9. The rSPV has been shown to elicit stable immune response by SNT.

10. Heterologous Prime-Boost vaccination regimen has been found to generate SN Titres for longer duration.

11. Potent Cell Mediated Immune response has been observed by Gamma Interferon ELISPOT assay.

12. Heterologous prime boost with simultaneous Protein and rSPV has been found to give good CMI response.

13. The utility of the rSPV as a marker vaccine has been demonstrated by amplification of GFP from blood samples after vaccination in sheep.

ACKNOWLEDGEMENTS Dr VVS Suryanarayana

Dr S Gopalakrishna

Dr C Ashok Kumar

Dr P Ravikumar

Dr G Nagarajan

Dr T Saravanan

Dr GR Reddy

Animal Care Staff

Mr VVV Rajkumar

Family and Friends