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Avian Influenza
A disease with a history
World Influenza Outbreaks
1934
Puerto Rico
H1N1
Cont. …
1995
Mexico
H5N2
Cont. …
1997
Hong Kong
H5N1 The potential of spreading H5N1 to human
was prevented by slaughtering all poultry in
Hong Kong in December 1997
World Influenza Outbreaks
2001
Hong Kong
H5N1 H5N1 reemerged again in Hong Kong in multiple
genotypes.
Controlled, again, by slaughtering all poultry in
Hong Kong.
World Influenza Outbreaks
2002
Hong Kong
H5N1 H5N1 reemerged again in Hong Kong in multiple
genotypes.
Controlled by slaughtering positive flocks and
infected poultry stalls in markets.
Hong Kong Story
Strategies to attempt to control the
emergence of highly pathogenic H5N1
viruses in Hong Kong poultry markets.
Hong Kong Story
 Slaughter of infected farms and live poultry
markets.
 All poultry in Hong Kong were slaughtered.1997
2002
 Birds in the markets and birds due to go to the
markets were slaughtered.
 No farms in Hong Kong were shown to be infected.
2001
Serological screening of each truckload of
poultry sold from local farms or entering
Hong Kong.
Removal of aquatic birds including ducks
and geese from the live poultry markets in
1998.
Quail was identified as the poultry species
that continued to reintroduce H5N1 and
H9N2 viruses into the Hong Kong poultry
markets and were banned.
Introduction of one “clean day” per
month when all poultry markets are
simultaneously empty and are cleaned.
What After Hong Kong Story
 These measures successfully removed the different H5N1
influenza virus genotypes from Hong Kong poultry markets.
 They have so far not prevented reemergence of H5N1
influenza viruses.
Cont. …
After the third reintroduction of
H5N1/02 influenza virus in Hong Kong,
the decision was made to investigate
the use of inactivated vaccine to
reduce the H5N1 virus load and inhibit
the further reemergence of H5N1
genotypes.
H5N2 … The First Use
Inactivated vaccine was successfully
used to control the H5N2 outbreak
in Mexico in 1995 (Villard and
Flores, 1997).
Cont. …
 The efficacy of commercially available
H5N2 influenza vaccine against H5N1/02
influenza viruses from Hong Kong should
be investigated.
 The only commercially available
inactivated H5 influenza vaccine is against
A/CK/Mexico/232/94 (H5N2).
ASSESSMENT
H5N2 … Immune Response
Amino acid sequence identity
between the HA of:
A/Ck/Mexico/232/94 (H5N2)
&
A/Ck/Hong Kong/86.3/02 (H5N1)
To evaluate the immune
response after single and
multiple doses of commercial
inactivated H5N2 influenza
vaccine.
89.3%
Study Design
 8-day-old chickens, were divided into 3 groups;
1. Vaccinated chickens with single dose of
A/CK/Mexico/232/94 (H5N2)
2. Unvaccinated in contact with the vaccinated chickens
3. Unvaccinated chickens
Evaluation
 Hemaggtutination inhibition (HI) titers:
1. A/CK/Hidalgo/232/94 (H5N2)
2. A/CK/HK/86.3/02 (H5N1)
Experiment 1
Profile:
Vaccinated group (19)
• Primary dose of vaccine at 8 day of age - challenged
Unvaccinated in contact (10)
• Not vaccinated - not challenged
Unvaccinated group (9)
• Not vaccinated - challenged
Challenge:
Aggressive challenge dose of 10 CLD50 of A/CK/HK/86.3/02
(H5N1).
Results
Mean HI Antibody Titer
H5N1 H5N2
Prechallenge Postchallenge Prechallenge Postchallenge
Vaccinated birds 20 53 100 150
Unvaccinated in contact - - - -
Unvaccinated - - - -
Deaths & Virus Shedding - 3 days post challenge
Deaths Tracheal shedding Cloaca shedding
Vaccinated birds 0/19 0/19 6/19
Unvaccinated in contact 0/10 0/10 1/10
Unvaccinated 6/9 0/3 1/3
Experiment 2
Profile:
Vaccinated group (10)
• Primary dose of vaccine at 8 day of age
• Booster dose at 28 days later – challenged.
Unvaccinated in contact (6)
• Not vaccinated – not challenged.
Unvaccinated group (9)
• Not vaccinated – challenged.
Challenge:
Aggressive challenge dose of 100 CLD50 of A/CK/HK/86.3/02
(H5N1).
Results
Mean HI Antibody Titer
H5N1 H5N2
Prechallenge Postchallenge Prechallenge Postchallenge
Vaccinated birds 333 337 391 257
Unvaccinated in contact - - - -
Unvaccinated - - - -
Deaths & Virus Shedding - 3 days post challenge
Deaths Tracheal shedding Cloaca shedding
Vaccinated birds 1/10 3/10 0/10
Unvaccinated in contact 3/6 3/6 2/6
Unvaccinated 9/9 NA NA
Experiment 3
Profile:
Vaccinated group (10)
• Primary dose of vaccine at 8 day of age
• Booster dose at 28 days later
Unvaccinated group (4)
• Nothing
Challenge:
Exposed to birds infected with 100 CLD50 H5N1
Results
Mean HI antibody titer
H5N1 H5N2
Prechallenge Postchallenge Prechallenge Postchallenge
Vaccinated birds 282 252 388 326
Unvaccinated - - - -
Deaths & Virus Shedding
Deaths Tracheal shedding Cloaca shedding
Vaccinated birds 0/10 1/10 0/10
Unvaccinated 3/4 0/1 0/1
CONCLUSION
Conclusion
 Thus commercial inactivated oil-emulsion A/CK/Mexico/
232/94 (H5N2) provides protection against challenge with low
doses of A/CK/HK/86.3/02 (H5N1) virus but did not prevent
virus shedding, which transmitted to contact birds.
 Despite high levels of HI antibody, the heavily challenged birds
were not fully protected, thus raising the question of lack of
genetic similarity between the vaccine and challenge virus.

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Avian Influenza History

  • 1. Avian Influenza A disease with a history
  • 4. Cont. … 1997 Hong Kong H5N1 The potential of spreading H5N1 to human was prevented by slaughtering all poultry in Hong Kong in December 1997
  • 5. World Influenza Outbreaks 2001 Hong Kong H5N1 H5N1 reemerged again in Hong Kong in multiple genotypes. Controlled, again, by slaughtering all poultry in Hong Kong.
  • 6. World Influenza Outbreaks 2002 Hong Kong H5N1 H5N1 reemerged again in Hong Kong in multiple genotypes. Controlled by slaughtering positive flocks and infected poultry stalls in markets.
  • 7. Hong Kong Story Strategies to attempt to control the emergence of highly pathogenic H5N1 viruses in Hong Kong poultry markets.
  • 8. Hong Kong Story  Slaughter of infected farms and live poultry markets.  All poultry in Hong Kong were slaughtered.1997 2002  Birds in the markets and birds due to go to the markets were slaughtered.  No farms in Hong Kong were shown to be infected. 2001
  • 9. Serological screening of each truckload of poultry sold from local farms or entering Hong Kong. Removal of aquatic birds including ducks and geese from the live poultry markets in 1998. Quail was identified as the poultry species that continued to reintroduce H5N1 and H9N2 viruses into the Hong Kong poultry markets and were banned. Introduction of one “clean day” per month when all poultry markets are simultaneously empty and are cleaned.
  • 10. What After Hong Kong Story  These measures successfully removed the different H5N1 influenza virus genotypes from Hong Kong poultry markets.  They have so far not prevented reemergence of H5N1 influenza viruses.
  • 11. Cont. … After the third reintroduction of H5N1/02 influenza virus in Hong Kong, the decision was made to investigate the use of inactivated vaccine to reduce the H5N1 virus load and inhibit the further reemergence of H5N1 genotypes.
  • 12. H5N2 … The First Use Inactivated vaccine was successfully used to control the H5N2 outbreak in Mexico in 1995 (Villard and Flores, 1997).
  • 13. Cont. …  The efficacy of commercially available H5N2 influenza vaccine against H5N1/02 influenza viruses from Hong Kong should be investigated.  The only commercially available inactivated H5 influenza vaccine is against A/CK/Mexico/232/94 (H5N2).
  • 15. H5N2 … Immune Response Amino acid sequence identity between the HA of: A/Ck/Mexico/232/94 (H5N2) & A/Ck/Hong Kong/86.3/02 (H5N1) To evaluate the immune response after single and multiple doses of commercial inactivated H5N2 influenza vaccine. 89.3%
  • 16. Study Design  8-day-old chickens, were divided into 3 groups; 1. Vaccinated chickens with single dose of A/CK/Mexico/232/94 (H5N2) 2. Unvaccinated in contact with the vaccinated chickens 3. Unvaccinated chickens
  • 17. Evaluation  Hemaggtutination inhibition (HI) titers: 1. A/CK/Hidalgo/232/94 (H5N2) 2. A/CK/HK/86.3/02 (H5N1)
  • 18. Experiment 1 Profile: Vaccinated group (19) • Primary dose of vaccine at 8 day of age - challenged Unvaccinated in contact (10) • Not vaccinated - not challenged Unvaccinated group (9) • Not vaccinated - challenged Challenge: Aggressive challenge dose of 10 CLD50 of A/CK/HK/86.3/02 (H5N1).
  • 19. Results Mean HI Antibody Titer H5N1 H5N2 Prechallenge Postchallenge Prechallenge Postchallenge Vaccinated birds 20 53 100 150 Unvaccinated in contact - - - - Unvaccinated - - - - Deaths & Virus Shedding - 3 days post challenge Deaths Tracheal shedding Cloaca shedding Vaccinated birds 0/19 0/19 6/19 Unvaccinated in contact 0/10 0/10 1/10 Unvaccinated 6/9 0/3 1/3
  • 20. Experiment 2 Profile: Vaccinated group (10) • Primary dose of vaccine at 8 day of age • Booster dose at 28 days later – challenged. Unvaccinated in contact (6) • Not vaccinated – not challenged. Unvaccinated group (9) • Not vaccinated – challenged. Challenge: Aggressive challenge dose of 100 CLD50 of A/CK/HK/86.3/02 (H5N1).
  • 21. Results Mean HI Antibody Titer H5N1 H5N2 Prechallenge Postchallenge Prechallenge Postchallenge Vaccinated birds 333 337 391 257 Unvaccinated in contact - - - - Unvaccinated - - - - Deaths & Virus Shedding - 3 days post challenge Deaths Tracheal shedding Cloaca shedding Vaccinated birds 1/10 3/10 0/10 Unvaccinated in contact 3/6 3/6 2/6 Unvaccinated 9/9 NA NA
  • 22. Experiment 3 Profile: Vaccinated group (10) • Primary dose of vaccine at 8 day of age • Booster dose at 28 days later Unvaccinated group (4) • Nothing Challenge: Exposed to birds infected with 100 CLD50 H5N1
  • 23. Results Mean HI antibody titer H5N1 H5N2 Prechallenge Postchallenge Prechallenge Postchallenge Vaccinated birds 282 252 388 326 Unvaccinated - - - - Deaths & Virus Shedding Deaths Tracheal shedding Cloaca shedding Vaccinated birds 0/10 1/10 0/10 Unvaccinated 3/4 0/1 0/1
  • 25. Conclusion  Thus commercial inactivated oil-emulsion A/CK/Mexico/ 232/94 (H5N2) provides protection against challenge with low doses of A/CK/HK/86.3/02 (H5N1) virus but did not prevent virus shedding, which transmitted to contact birds.  Despite high levels of HI antibody, the heavily challenged birds were not fully protected, thus raising the question of lack of genetic similarity between the vaccine and challenge virus.

Editor's Notes

  1. In a third experiment, birds that had received two doses of vaccine were challenged under conditions more akin to field conditions by being exposed to dying birds that had been inoculated with 100 CLD50 of A/CK/HK/86.3/02 (H5N1). Under these conditions, none of the vaccinated birds developed disease signs or died, but one shed virus (from the trachea).