COMPARISON OF THE ADAPTIVE IMMUNE

RESPONSES INDUCED BY FMD-VACCINES

FORMULATED WITH 146S AND 75S PARTICLES IN MICE

AND CATTLE

Dra. María Cruz MiragliaPost-doctoral fellow

Institute of Virology and Technical Innovations. INTA-CONICET. Buenos Aires.

OBJECTIVETo compare the immunogenicity of FMDV natural empty capsids (75S) vs. whole viral

particles (146S) from the same virus strain.

✓ 146S and 75S antigens were purified from the same inactivated antigenicpreparation by means of a 15%-45% discontinuous sucrose gradient.

✓ Single oil emulsion vaccines were formulated using the purified 75S and146S particles from the A24/Cruzeiro strain.

✓ Both oil-vaccines contained 1.5 µg/ml and were used to immunize miceand cattle.

EXPERIMENTAL DESIGN

g/ml

Purificación de cápsides vacías naturales

146S75S

256 128 64

SDS-PAGE 10%

VP0

VP1VP2+ VP3

Antigen purification

• Concentration of 146S particles was estimated byRNA spectrophotometry (260 nm)

• Concentration of 75S particles was estimated by using 146S as a reference

• Yields of 146S particles were approximately 6 times higher than those of the 75S particles

43

34

32Pure 1/2 1/4 1/8 1/16

26

111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596061626364656667686970710.0

0.5

1.0

1.5

2.0

2.5 260 nm

490 nm antigen-ELISA

146S

75S 12S

Fraction

Ab

so

rban

ce

7 14 21 26 0

C57bl/6

n=8 animals/group

NET

146S

75S

1 3 7

0 1 2 3 4 5 6 70

50

100

150

NET

75S

146S

Days post-infection

Su

rviv

al (%

)

Challenge 1x106 UFP/200 µl i.p.146S or 75S vaccines

(0.3 µg/dose)

Experimental challenge in mice

Experimental Design in Mice

• Animals vaccinated with 146S or 75S particles survived to experimental challengewhile all animals in the control group (NET) died between 3 and 4 dpi.

serum samples

0 7 14 210

2

4

6

27 30 33

NET75S146S

**** * *

Days

A24/C

ruzeir

o

EL

ISA

Ig

G (

Lo

g1

0)

21dpv

75S 146S 0

2

4

6***

p=0,0007

A24/C

ruzeir

o

EL

ISA

Ig

G (

Lo

g1

0)

1dpi

75S 146S0

2

4

6 *p=0,0159

A24/C

ruzeir

o

EL

ISA

Ig

G (

Lo

g1

0)

3 dpi

75S 146S0

2

4

6 *p=0,0195

A24/C

ruzeir

o

EL

ISA

Ig

G (

Lo

g1

0)

26 dpv

75S 146S0

2

4

6 *p=0,0159

A24/C

ruzeir

o

EL

ISA

Ig

G (

Lo

g1

0)

Challenge

• 146S particle vaccines induced significantlyhigher FMDV-specific IgG titers than 75Svaccines starting at 21 dpv

26dpv

146S 75S0

2

4

6

SN

V t

iter

(Lo

g1

0)

3 dpi

146S 75S0

2

4

6

SN

V t

iter

(Lo

g1

0)

7 dpi

146S 75S0

2

4

6

SN

V t

iter

(Lo

g1

0)

Anti-FMDV IgG and Neutralizing Antibody Titers

• Mean neutralizing antibodies titers in 75Sgroup were 10 to 30 times lower than those in146S vaccinated group

FMDV-specific IgG1

0 7 14 210

2

4

6

27 30 33

NET

75S

146S

Days

A24/C

ruzeir

o

EL

ISA

Ig

G1 (

Lo

g1

0)

FMDV-specific IgG2a

0 7 14 210

2

4

6

27 30 33

NET75S

146S

****

** *

Days

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

21 dpv

75S 146S0

2

4

6

**p=0,0014

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

14 dpv

75S 146S0

2

4

6

**p=0,0035

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

26 dpv

75S 146S0

2

4

6*

p=0,0159

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

3 dpi

75S 146S0

2

4

6

*p=0,0357

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

7 dpi

75S 146S0

2

4

6 *p=0,0159

A24/C

ruzeir

o

EL

ISA

Ig

G2

a (

Lo

g1

0)

Challenge

Challenge

Anti-FMDV IgG Isotype Antibody Titers

• Animals vaccinated with 146S and 75Sparticles do not show any differences asregard IgG1 antibody titers

• As of 14 dpv, IgG2a titers were higher in the146S vaccinated group than in the 75Svaccinated group

7 14 21 28 0

n=4 animals/group

3 µg/dose

146S

75S

45 70 91

Commercial tetravalent

vaccine (70 dpv)

146S or 75S vaccines(3 µg/dose)

Calves <8 months-old

without colostral

antibodies.

Experimental Design in Cattle

whole bloodsamples

serum samples

Days post-vaccination

IFN-gamma

Serological assessments: FMDV-specific IgG antibodies and Neutralizing antibodies

0 20 40 60 80 1000

1

2

3

4

5 146s75s

* **

Protective Threshold

EPP 75%#

Days post vaccination

A24/C

ruzeir

o

LP

-EL

ISA

tit

er

(Lo

g1

0)

28 dpv

75S 146S0

1

2

3

4

5

*p=0.0286

A24/C

ruzeir

o

LP

-EL

ISA

tit

er

(Lo

g1

0)

45 dpv

75S 146S0

1

2

3

4

5

*p=0.0286

A24/C

ruzeir

o

LP

-EL

ISA

tit

er

(Lo

g1

0)

70 dpv

75S 146S0

1

2

3

4

5

*p=0.0286

A24/C

ruzeir

o

LP

-EL

ISA

tit

er

(Lo

g1

0)

SNV

0 20 40 60 80 1000

1

2

3

4

5146S

75S

***

Protective Threshold

EPP 75% ##

Days post vaccination

SN

V t

iter

(Lo

g1

0)

21 dpv

75S 146S0

1

2

3

4

*p=0.0286

SN

V t

iter

(Lo

g1

0)

28 dpv

75S 146S0

1

2

3

4

*p=0.0286

SN

V t

iter

(Lo

g1

0)

45 dpv

75S 146S0

1

2

3

4

*p=0.0286

SN

V t

iter

(Lo

g1

0)

Commercial tetravalent vaccine

Commercial tetravalent vaccine

Cattle vaccinated with 146S particles showed higher total antibody titers than

those immunized with 75S particles

between 28 and 70 dpv.

Anti-FMDV Total and Neutralizing Antibody Titers

# Maradei E, 2008.## PANAFTOSA, 1994.

Cattle vaccinated with 75S vaccine has undetectable levels

of NAb

146S

0 50 100

2

3

4

5

IgMIgG1

IgG2

Days post vaccination

A24/C

ruzeir

o

EL

ISA

tit

er

(Lo

g10)

75S

0 50 100

2

3

4

5

IgMIgG1

IgG2

Days post vaccination

A24/C

ruzeir

o

EL

ISA

tit

er

(Lo

g10)

21dpv

75S 146S0

1

2

3

4*

p=0.0286

A24/C

ruzeir

o

EL

ISA

Ig

G1 t

iter

(Lo

g1

0)

28 dpv

75S 146S0

1

2

3

4*

p=0.0286

A24/C

ruzeir

o

EL

ISA

Ig

G1 t

iter

(Lo

g1

0)

45 dpv

75S 146S0

1

2

3

4 *p=0.0286

A24/C

ruzeir

o

EL

ISA

Ig

G1 t

iter

(Lo

g1

0)

IgG1

• Animals vaccinated with 75S particles didnot induce IgG2 and showed significantlylower IgG1 levels compared to animalsvaccinated with 146S particles

Anti-FMDV Isotype Antibody Titers

Commercial tetravalent vaccine

Commercial tetravalent vaccine

Vaccine

PWM NET

PWM NET

PWM NET

PWM NET

PWM NET

PWM NET

Stimulating antigen

• Stimulation with 75S particleswas unable to promote in vitrocell-mediated immune responses

• The onset of the antigen-specificIFN-γ production was delayed in75S vaccinated cattle

FMDV-specific Cellular Responses

0 7 142128457091 0 7 142128457091 0 7 142128457091 0 7 1421284570910

1.0105

2.0105

3.0105

75S vaccine 146S vaccine 75S vaccine 146S vaccine

75S 146S

DPV

Experimental group

In vitro stimulation

IFN

- (

pg

/ml)

✓ Immunological differences also include lower levels of totalantibodies and changes in the induced IgG isotype profiles, bothin mice and cattle

✓ Further studies are needed to assess the protective capacity ofthe empty capsids in target species

✓ Natural empty capsids failed to induce antigen-specific in vitroIFN-gamma responses in vaccinated cattle

✓ Vaccination with natural empty capsids induced very low orundetectable levels of NAb titers in mice and cattle

Dra. Sabrina CardilloDra. Eliana SmitsaartDra. Ana Maria Espinosa

Dr. Mariano Pérez FilgueiraDra. Alejandra Capozzo

Dr. Bucafusco DaniloMSc. Di Giacommo, SebastiánLic. Schammas Juan ManuelDra. Barrionuevo, FlorenciaTec. Ayude, Andrea

PID 2013 N°022

Thank you!

0

1

2

3

4

0.0

0.5

1.0

1.5

260 nm

280 nm 490 nmO

D 2

60/2

80 n

m OD

490 n

m

0 20 40 60 80 100 120 1400

1

2

3

4

5

146S

75S

EPP 75%

Days post vaccination

SN

V t

iter

(Lo

g10)

0 10 20 300

1

2

3

4

5

146s

75 s

EPP 75%

Days post vaccination

A24/C

ruzeir

o

LP

-EL

ISA

tit

er

(Lo

g1

0)

0 7 14212863 0 7 14212863 0 7 14212863 0 7 142128630

1.0105

2.0105

3.0105

4.0105

IFN

- p

g/m

l

75S vaccine 146S vaccine 75S vaccine 146S vaccine

146S

DPV

Experimental group

In vitro stimulation75S

*

Cattle vaccinated with 146S or 75S (10 µg/dose)