4TH MERIAL FORUM

HAVE WE GOT PCVD &

SWINE INFLUENZA UNDER

CONTROL?

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2

swine influenza virus in pigs

Merial Swine Forum, Berlin 1st of June, 2012 Karen van der Meulen

• Introduction and aims

• Materials and methods

• Results

• Conclusions

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2 swine influenza virus in pigs

• Introduction and aims

• Materials and methods

• Results

• Conclusions

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2 swine influenza virus in pigs

Introduction: swine influenza virus

• Swine influenza virus (SIV)

• Family Orthomyxoviridae

• Influenza A virus

• 8 single stranded RNA segments

• Encoding different proteins

hemagglutinin (HA), 16 types

neuraminidase (NA), 9 types

internal proteins (PB1, PB2, PA, NP, NS and M)

Introduction: swine influenza virus

neuraminidase (NA) N1-N9

hemagglutinin (HA) H1-H16

Introduction: swine influenza virus

• Swine influenza virus (SIV)

• Family Orthomyxoviridae

• Influenza A virus

• 8 single stranded RNA segments

• Encoding different proteins

•Subtyped based on the type of HA and NA:

H1N1, H3N2, H5N1, …

•Nomenclature:

A/swine/Belgium/1/98, A/California/04/09

Introduction: swine influenza virus

• Three subtypes enzootic in pigs throughout the world:

H1N1, H3N2, H1N2

• Antigenic and genetic differences between regions

• SIVs evolve at much slower rate than their human

counterparts, but some extent of antigenic drift may

occur

• Genetic reassortment occurs frequently in pigs

avian-like

H1N2 1994

H1N1 1979

reassortant

Since 2009: pandemic (p)H1N1 humans and swine

Prevalence?

Chile/83

duck/77

H3N2 1984

reassortant Hong Kong/68

Introduction: swine influenza virus

Introduction: swine influenza virus

Major cause of acute respiratory disease in pigs, although

most infections are subclinical

Depression, fever, laboured and abdominal breathing

Pneumonia

Vaccination is the most effective means of preventing swine influenza

Introduction: swine influenza virus

Bivalent SIV vaccines used in Europe

Name

Gripovac®

Respiporc®

Flu

Suvaxyn®

Flu

Company

Merial

IDT

Pfizer AH

Virus strains

New Jersey/8/76

Port Chalmers/1/73

Sw/Belgium/230/92

Sw/Belgium/220/92

Sw/Netherlands/25/80

Port Chalmers/1/73

Adjuvant

Oil in water

Oil in water

Oil + AlOH

Virus subtype

H1N1

H3N2

H1N1

H3N2

H1N1

H3N2

• No/minimal cross-reactive serum hemagglutination

inhibiting (HI) antibodies against H1N2

• No protection against challenge (Van Reeth et al., Vet. Record 2003)

Introduction: swine influenza virus

H1N2 1994

H1N1 1979

pH1N1 2009

Percentage amino acid (aa) identity between H1 SIV lineages in Europe

70.5%

72%

70%

14 aa changes in antigenic sites

28 aa changes

Introduction: swine influenza virus

Trivalent SIV vaccine used in Europe (since 2009)

Name

Gripovac®3

/Respiporc®

Flu3

Company

Merial

Virus strains

Sw/Haselünne/2671/2003

Sw/Bakum/1769/3002

Sw/Bakum/1832/2000

Adjuvant

Carbomer

Virus subtype

H1N1

H3N2

H1N2

First trivalent vaccine containing all

three SIV subtypes including H1N2

Aims

1. To examine the protective properties of

Gripovac®3/Respiporc®Flu3 against intratracheal

challenge with a recent H1N2 SIV

1. To study protection upon intranasal and aerosol

challenge, more closely simulating the natural course

of infection

• Introduction and aims

• Materials and methods

• Results

• Conclusions

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2 swine influenza virus in pigs

Materials and methods

Animals:

•influenza-negative pigs, 6-weeks old

•36 control and 36 vaccinated

Vaccine:

•Gripovac®3 /Respiporc® Flu3 (i.m.)

•2 x with 3-week interval

Challenge virus:

•Sw/Gent/102/07 (H1N2)

•95.5% amino acid identity with HA of vaccine strain

Materials and methods

0 3 6 (weeks)

Control

Vaccinated

/

Vaccination

2 ml IM

/

Vaccination

2 ml IM

Challenge

IT

IN

or aerosol

Challenge

IT

IN

or aerosol

Materials and methods

Control

Vaccinated

/

Vaccination

2 ml IM

/

Vaccination

2 ml IM

Challenge

IT

IN

or aerosol

Challenge

IT

IN

or aerosol

Sampling and euthanasia

Nasal swabs -> virus excretion

Lung -> virus titres / lesions

Nasal mucosa -> virus titres

Sampling and euthanasia

Nasal swabs -> virus excretion

Lung -> virus titres / lesions

Nasal mucosa -> virus titres

0 3 6 7 (weeks)

Clinical monitoring and blood sampling throughout the study

• Introduction and aims

• Materials and methods

• Results

• Conclusions

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2 swine influenza virus in pigs

Results: Vaccination with Gripovac®3 /Respiporc® Flu3

Antibody response in vaccinated pigs

0

20

40

60

80

100

120

Primary vaccination(V1)

1 week post V2 2 weeks post V2 3 weeks post V2(challenge)

H1N1

H3N2

H1N2 - vaccine

H1N2 - challenge

HI a

nti

bo

dy

titr

e (g

eom

etri

c m

ean

) N

o. p

os.

23 25 25 10 36 36 36 36 36 36 36 36 36 36 36 32

Results: Vaccination with Gripovac®3 /Respiporc® Flu3

Antibody response in vaccinated pigs

0

20

40

60

80

100

120

Primary vaccination(V1)

1 week post V2 2 weeks post V2 3 weeks post V2(challenge)

H1N1

H3N2

H1N2 - vaccine

H1N2 - challenge

HI a

nti

bo

dy

titr

e (g

eom

etri

c m

ean

) N

o. p

os.

23 25 25 10 36 36 36 36 36 36 36 36 36 36 36 32

• Antibodies vaccine strains -> vast majority pigs

Results: Vaccination with Gripovac®3 /Respiporc® Flu3

Antibody response in vaccinated pigs

0

20

40

60

80

100

120

Primary vaccination(V1)

1 week post V2 2 weeks post V2 3 weeks post V2(challenge)

H1N1

H3N2

H1N2 - vaccine

H1N2 - challenge

HI a

nti

bo

dy

titr

e (g

eom

etri

c m

ean

) N

o. p

os.

23 25 25 10 36 36 36 36 36 36 36 36 36 36 36 32

• Antibodies vaccine strains -> vast majority pigs

• Antibodies challenge strain -> majority of pigs but lower titres

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean clinical scores upon challenge

In non-vaccinated control pigs:

• Most pronounced clinical signs after aerosol challenge

• Intranasal inoculation largely subclinical

• Lung lesion most extensive upon aerosol challenge

0

4

8

12

16

20

0 1 2 3 4 5

0

4

8

12

16

20

0 1 2 3 4 5

0

4

8

12

16

20

0 1 2 3 4 5

Mea

n c

linic

al s

core

Intratracheal Intranasal Aerosol

Days post challenge

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean clinical scores upon challenge

0

4

8

12

16

20

0 1 2 3 4 5

0

4

8

12

16

20

0 1 2 3 4 5

0

4

8

12

16

20

0 1 2 3 4 5

Mea

n c

linic

al s

core

Intratracheal Intranasal Aerosol

Days post challenge

In vaccinated pigs:

• Fewer animals show clinical signs

• Clinical signs are milder

* * * *

* * *

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean virus titres in the left lung

In non-vaccinated control pigs:

• High virus titres in the lung for all three challenge methods

• Highest titres after aerosol inoculation

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/g

r)

Days post challenge

Intratracheal Intranasal Aerosol

0

2

4

6

8

10

1 3 5

0

2

4

6

8

10

1 3 50

2

4

6

8

10

1 3 5

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean virus titres in the left lung

In vaccinated pigs:

• Reduction in the extent of virus replication for all three

challenge methods

• Most pronounced protective effect for aerosol challenge

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/g

r)

Days post challenge

Intratracheal Intranasal Aerosol

* *

* *

*

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/g

r)

Days post challenge

Intratracheal Intranasal Aerosol

0

2

4

6

8

10

1 3 5

0

2

4

6

8

10

1 3 50

2

4

6

8

10

1 3 5

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean virus titres in the nasal mucosa

In vaccinated pigs:

• Reduction in the extent of virus replication for all three

challenge methods

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/g

r)

Days post challenge

Intratracheal Intranasal Aerosol

* *

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/g

r)

Days post challenge

Intratracheal Intranasal Aerosol

0

2

4

6

8

10

1 3 50

2

4

6

8

10

1 3 5

0

2

4

6

8

10

1 3 5

*

* *

* *

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean nasal virus excretion

In non-vaccinated control pigs:

• Nasal virus excretion for all three challenge methods

• Most consistent after intranasal inoculation

• Delayed nasal excretion after intratracheal inoculation

0

2

4

6

8

10

0 1 2 3 4 5

0

2

4

6

8

10

0 1 2 3 4 5

0

2

4

6

8

10

0 1 2 3 4 5

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/m

g)

Days post challenge

Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2

Mean nasal virus excretion

In vaccinated pigs:

• Reduced nasal virus excretion for all three challenge methods

• Delayed nasal virus excretion upon intratracheal challenge likely

biases protective response

0

2

4

6

8

10

0 1 2 3 4 5

0

2

4

6

8

10

0 1 2 3 4 5

0

2

4

6

8

10

0 1 2 3 4 5

Mea

n v

iru

s ti

tre

(lo

g 10

TC

ID5

0/m

g)

Days post challenge

* * * *

*

* *

*

*

• Introduction and aims

• Materials and methods

• Results

• Conclusions

Efficacy of Gripovac®3/Respiporc®Flu3 against challenge with a recent H1N2 swine influenza virus in pigs

Conclusions

Gripovac®3/Respiporc®Flu3 induces a partial clinical and

virological protection against challenge with a recent H1N2

• Clinical signs

• Virus titres in the lung and upper respiratory tract

• Nasal virus excretion

Conclusions

Gripovac®3/Respiporc®Flu3 induces a partial clinical and

virological protection against challenge with a recent H1N2

• Clinical signs

• Virus titres

• Nasal virus excretion

Protection at “pig-level”

Conclusions

Gripovac®3/Respiporc®Flu3 induces a partial clinical and

virological protection against challenge with a recent H1N2

Protection at “farm-level”

• Clinical signs

• Virus titres

• Nasal virus excretion

Conclusions

Currently no requirement to test nasal virus

excretion during marketing authorization

Conclusions

• Easy to determine

• No euthanasia required

But …

• Importance of the route of challenge

-> location of primary virus deposition

Currently no requirement to test nasal virus

excretion during marketing authorization

Prof. Dr. K. Van Reeth

P. Elskens

L. Sys

N. Dennequin

M. Bauwens

Z. Van den Abeele Dr. M. Bublot

Dr. T. Vila

Dr. F. Joisel

Dr. T. Meyns

Dr. E. Mundt

Dr. R. Dürrwald

Dr. M. Schlegel