This document discusses a study that evaluated the efficacy of the Gripovac®3/Respiporc®Flu3 swine influenza vaccine against a recent H1N2 swine influenza virus challenge in pigs. The study involved vaccinating pigs with the trivalent vaccine and then challenging them with the H1N2 virus via intratracheal, intranasal, or aerosol exposure. Results showed that vaccination induced antibody responses against the vaccine strains in the vast majority of pigs and provided protection upon challenge as evidenced by reduced virus shedding and lung lesions compared to control pigs. The study demonstrates that the trivalent vaccine can provide protection against currently circulating H1N2 swine influenza viruses.

1. 4TH MERIAL FORUM
HAVE WE GOT PCVD &
SWINE INFLUENZA UNDER
CONTROL?

2. Efficacy of Gripovac®3/Respiporc®Flu3
against challenge with a recent H1N2
swine influenza virus in pigs
Merial Swine Forum, Berlin Karen van der Meulen 1st of June, 2012

3. 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

4. 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

5. 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)

6. Introduction: swine influenza virus
hemagglutinin (HA)
H1-H16
neuraminidase (NA)
N1-N9

7. 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

8. 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

9. Introduction: swine influenza virus
H1N1
1979
duck/77 avian-like Since 2009:
pandemic (p)H1N1
H3N2 humans and swine
1984
Hong Kong/68 reassortant
Prevalence?
H1N2
1994
Chile/83 reassortant

10. Introduction: swine influenza virus
Major cause of acute respiratory disease in pigs, although
most infections are subclinical
Depression, fever, laboured and Pneumonia
abdominal breathing
Vaccination is the most effective means of
preventing swine influenza

11. Introduction: swine influenza virus
Bivalent SIV vaccines used in Europe
Name Company Virus strains Virus subtype Adjuvant
Gripovac® Merial New Jersey/8/76 H1N1 Oil in water
Port Chalmers/1/73 H3N2
Respiporc® IDT Sw/Belgium/230/92 H1N1 Oil in water
Flu Sw/Belgium/220/92 H3N2
Suvaxyn® Pfizer AH Sw/Netherlands/25/80 H1N1 Oil + AlOH
Flu Port Chalmers/1/73 H3N2
• No/minimal cross-reactive serum hemagglutination
inhibiting (HI) antibodies against H1N2
• No protection against challenge
(Van Reeth et al., Vet. Record 2003)

12. Introduction: swine influenza virus
Percentage amino acid (aa) identity between H1 SIV
lineages in Europe
H1N1 72%
1979 14 aa changes in
antigenic sites
70.5% pH1N1
28 aa changes 2009
70%
H1N2
1994

13. Introduction: swine influenza virus
Trivalent SIV vaccine used in Europe (since 2009)
Name Company Virus strains Virus subtype Adjuvant
Gripovac®3 Merial Sw/Haselünne/2671/2003 H1N1 Carbomer
/Respiporc® Sw/Bakum/1769/3002 H3N2
Flu3 Sw/Bakum/1832/2000 H1N2
First trivalent vaccine containing all
three SIV subtypes including H1N2

14. 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

15. 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

16. 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

17. Materials and methods
0 3 6 (weeks)
Control / / Challenge
IT
IN
or aerosol
Vaccinated Vaccination Vaccination Challenge
2 ml IM 2 ml IM IT
IN
or aerosol

18. Materials and methods
0 3 6 7 (weeks)
Control / / Challenge Sampling and euthanasia
IT Nasal swabs -> virus excretion
IN Lung -> virus titres / lesions
or aerosol Nasal mucosa -> virus titres
Vaccinated Vaccination Vaccination Challenge Sampling and euthanasia
2 ml IM 2 ml IM IT Nasal swabs -> virus excretion
IN Lung -> virus titres / lesions
or aerosol Nasal mucosa -> virus titres
Clinical monitoring and blood sampling throughout the study

19. 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

20. Results: Vaccination with Gripovac®3 /Respiporc® Flu3
Antibody response in vaccinated pigs
120
HI antibody titre (geometric mean)
100
80 H1N1
60 H3N2
H1N2 - vaccine
40
H1N2 - challenge
20
0
Primary vaccination 1 week post V2 2 weeks post V2 3 weeks post V2
(V1) (challenge)
No. pos.
23 25 25 10 36 36 36 36 36 36 36 36 36 36 36 32

21. Results: Vaccination with Gripovac®3 /Respiporc® Flu3
Antibody response in vaccinated pigs
120
HI antibody titre (geometric mean)
100
80 H1N1
60 H3N2
H1N2 - vaccine
40
H1N2 - challenge
20
0
Primary vaccination 1 week post V2 2 weeks post V2 3 weeks post V2
(V1) (challenge)
No. pos.
23 25 25 10 36 36 36 36 36 36 36 36 36 36 36 32
• Antibodies vaccine strains -> vast majority pigs

22. Results: Vaccination with Gripovac®3 /Respiporc® Flu3
Antibody response in vaccinated pigs
120
HI antibody titre (geometric mean)
100
80 H1N1
60 H3N2
H1N2 - vaccine
40
H1N2 - challenge
20
0
Primary vaccination 1 week post V2 2 weeks post V2 3 weeks post V2
(V1) (challenge)
No. pos.
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

23. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean clinical scores upon challenge
20
Intratracheal 20
Intranasal 20 Aerosol
16 16 16
Mean clinical score
12 12 12
8 8 8
4 4 4
0 0 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Days post challenge
In non-vaccinated control pigs:
• Most pronounced clinical signs after aerosol challenge
• Intranasal inoculation largely subclinical
• Lung lesion most extensive upon aerosol challenge

24. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean clinical scores upon challenge
20
Intratracheal 20
Intranasal 20 Aerosol
16 16 16
Mean clinical score
12 12 12
8 8 8
4 4 4
*
* *
* * * *
0 0 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Days post challenge
In vaccinated pigs:
• Fewer animals show clinical signs
• Clinical signs are milder

25. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean virus titres in the left lung
10
Intratracheal 10
Intranasal 10
Aerosol
Mean virus titre (log10 TCID50/gr)
8 8 8
6 6 6
4 4 4
2 2 2
0 0 0
1 3 5 1 3 5 1 3 5
Days post challenge
In non-vaccinated control pigs:
• High virus titres in the lung for all three challenge methods
• Highest titres after aerosol inoculation

26. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean virus titres in the left lung
10
Intratracheal 10
Intranasal 10
Aerosol
Mean virus titre (log10 TCID50/gr)
8 8 8 *
*
6 6 6
4 4 4
*
2 * 2 2 *
0 0 0
1 3 5 1 3 5 1 3 5
Days post challenge
In vaccinated pigs:
• Reduction in the extent of virus replication for all three
challenge methods
• Most pronounced protective effect for aerosol challenge

27. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean virus titres in the nasal mucosa
10
Intratracheal 10
Intranasal 10
Aerosol
Mean virus titre (log10 TCID50/gr)
8 8 8
6 6 6
*
4 4 * 4
2
* * 2 2
* * *
0 0 0
1 3 5 1 3 5 1 3 5
Days post challenge
In vaccinated pigs:
• Reduction in the extent of virus replication for all three
challenge methods

28. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean nasal virus excretion
10 10 10
Mean virus titre (log10 TCID50/mg)
8 8 8
6 6 6
4 4 4
2 2 2
0 0 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Days post challenge
In non-vaccinated control pigs:
• Nasal virus excretion for all three challenge methods
• Most consistent after intranasal inoculation
• Delayed nasal excretion after intratracheal inoculation

29. Results: Protection of Gripovac®3 /Respiporc® Flu3 against H1N2
Mean nasal virus excretion
10 10 10
Mean virus titre (log10 TCID50/mg)
8 8 8
6 * * 6
6
* *
4
* * * 4 4
*
*
2 2 2
0 0 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Days post challenge
In vaccinated pigs:
• Reduced nasal virus excretion for all three challenge methods
• Delayed nasal virus excretion upon intratracheal challenge likely
biases protective response

30. 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

31. 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 

32. 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”

34. 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
“farm-level”

35. Conclusions
Currently no requirement to test nasal virus
excretion during marketing authorization

36. Conclusions
Currently no requirement to test nasal virus
excretion during marketing authorization
• Easy to determine
• No euthanasia required
But …
• Importance of the route of challenge
-> location of primary virus deposition

37. 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