Development of Influenza
Vaccines for Pandemic
Preparedness in Taiwan
Ih-Jen Su
National Institute of Infectious Diseases and Vaccinology
National Health Research Institutes (NHRI)
Zhunan, Taiwan
Nov. 2014
Influenza virus
 Orthomyxoviridae family
Negative-sense
ssRNA
Neuraminidase (NA)
Hemagglutinin (HA)
http://www.itqb.unl.pt/labs/protein-modelling/activities/haemagglutinin
Nature Reviews Microbiology 6, 143-155 (February 2008)
Distribution of HA Receptor in the Epithelium of
Upper and Lower Respriatory Tracts, and Plays An
Important Role of Pandemic Influenza Transmission
EVOLUTION OF NEW INFLUENZA
VIRUS
- cross infection of human & avian
influenza in pigs
- leading to reassortment of genes and
new
strains
Transmission of avian
influenza virus by stool
Transmission of human
flu to pigs by aerosol
New flu virus
Spread
by
aerosol
Mechanism for the Transmission of Pandemic
Influenza A Virus from Poultry to Humans
Nat Rev Microbiol. 2006 Nov;4(11):857-64
Postulated Evolution of Influenza A Virus Pandemic Strains
Adapted from: BR Murphy & RG Webster “Orthomyxoviruses” in Fields Virology, 3rd Edition Lippincott-Raven Publishers
1889
H2N2
1900
H3N8
?
1918
H1N1
1957
H2N2
1968
H3N2
1977
H1N1
Spanish
Asian
Hong Kong
Russian
?
5
1997,2003H5N1
6
H5N1
8?
3
1918 outbreak of H1N1 in New York H1N1
H2N2
2
H3N? 1997 outbreak of H5N1 in Hong Kon
2003- Vietnam, China, Indonesia---
Global Circulation of H3N2 Influenza Virus: the majority of new flu
strain originate in China
Flu Epidemiology in Taiwan 1-2 yrs Ahead of the WHO
Vaccine Strain
Fig 1. Evolutionarily leading and trailing regions.
Fig 2. Schematic of the dominant seeding
hierarchy of seasonal influenza A (H3N2) viurses.
Shih SR, J Clin Microbiol, 2005;
Derek Smith, et al. Science 2008
Epidemiology of H5N1 Influenza
as of January, 2008
世界衛生組織，國際動物組織
2009 H1N1 學校停課情形
2000
the number of suspended class
停課班級數
展開校園集
NISIV
中接種作業
1500
1000
500
0
9/7 9/14 9/21 9/28 10/5 10/1210/1910/2611/2 11/9 11/1611/2311/3012/7 12/1412/2112/28 1/4
Date
10
Expectations for an Influenza Risk Assessment
Increasing Risk
Viruses - Highest Priority
Viruses - Increasing Priority
Viruses - Increasing Priority
Lower Priority
High Ag Yield
Reassortants,
Lab Studies
Safety Studies
Production
Scale-Up
Phase I
Clinical
Vaccine Development
Courtesy of Dr. Ruben Donis
The location of low pathogenic avian influenza H5N2
outbreaks in poultry farms in 2004 in Taiwan
Detection of avian
influenza H5N2 virus in
Taiwan, 2008
An
H5N2 influenza virus
was isolated from an
apparently healthy chicken in
a layer farm in Kaohsiung
county in October 2008.
All of the 18,000 chickens
were culled.
No virus has been isolated
from birds of 146 farms in 3km radius around the farm,
in extensive surveillance
until September 2009.
Courtesy of Taiwan AHRI
13
Recent HPAI H5N2 Outbreaks in Jan~ May 1012
•Highly pathogenic
avian influenza (HPAI)
H5N2 virus caused
outbreaks in 5
chicken farms and 1
slaughterhouse in
2012.
Changhua County
Yunlin County
Tainan City
Pingtung
Courtesy of Taiwan AHRI
Seroprevalence of HI antibody against
H5N2 avian influenza in healthy adults
point at 1:80
TableCutoff
3 Seroprevalence
of HI titers against H5N2 avian influenza
negative
positive
Group
P value Odds ratio
n
%
n
%
Live poultry vendors
325
97.00
10
3.00
0.001
8.85
Poultry farmers
329
98.20
6
1.80
0.057
5.24
Non-poultry workers
575
99.70
2
0.30
*cut point at 80 of HI titers
Sero-positive rate: 2.4% (16/670)
• combine live poultry vendors and poultry farmers together
15
Unpublished data and confidential (courtesy of Taiwan CDC)
Expert Committee
新型動物流感研討會結論 (13 Nov 2012)
•應加強整合台灣政府各農業相關機構與產業界之
合作，以增進動物流感疫情監測效率與準確度。
•人類流感與動物流感之病毒分離、基因序列及血
清學分析結果應彼此交流分享，並進行風險評估
，找出可能具高危害性之流感病毒株進行研究。
•台灣地區的禽流感H5N2病毒已在地化，且已在家
禽業者造成不顯性傳染，因此應透過跨部會合作
，著手開發人用H5N2流感疫苗之準備工作。
Generation of Pandemic Influenza Vaccine
Seed Viruses
Plasmid Rescue - RG
Classical reassortment
RT-PCR
NA
PB2
PB1
PA
PA
NP
M
NS
Wt
Wt
+
HA
HA
Ab Selection
(1/28 or 1/256)
NA
Transfection
Vero cells
Cloning
Donor strain
6:2 reassortant
Vaccine
LPAI – BSL2
RT-PCR
Donor strain
Pathogenicity tests in
Chicken and ferrets
Mass Production
LPAI – BSL2
HPAI – BSL3
WHO consultation meeting: Feb and Sep
Development of Influenza H5N2 Human Vaccines for
Pandemic Preparedness in Taiwan
Milestones
Organization
Timeline
疾管局 / 農委會
1. Cross-reactive antibody titers between
3 months
H5N1 and H5N2 viruses (human sera)
疾管局 /農委會 / 國防部 3 months
2. Generation of ferret antisera
3. Generation of seed viruses
4. Pathogenicity tests of seed viruses
國衛院 / 農委會
6 months
國衛院 / 農委會/ 國防部 6 months
5. Generation and validation of virus bank
國衛院 / CRO (委外)
6. Process Development and clinical lot
國衛院 / 產業界
7. Potency test for product release
國衛院 / 藥管局
8. Protection study in ferrets
國衛院 / 國防部
9. Preclinical test (toxicology)
10. Clinical trial
國衛院 / CRO (委外)
國衛院 / 藥管局/ 產業界
Generation of Ferret Antisera
• Based on sequence analysis, 5 H5N2 viruses isolated
in 2003, 2008, 2010, and 2012 (2 strains) will be used
to generate ferret antisera.
• Each virus strain with two different virus titers (10^5 and
10^7 TCID50) will be used to infect two ferrets (total 10
ferrets).
• Virus shedding will be monitored for 7 days.
• At day 14 after 1st infection, sera will be collected for
measuring sera neutralizing antibody titers in NHRI.
• If the ferrets don’t develop high neutralizing antibody
titers after 1st infection, 2nd infection will be given at day
21 and sera will be collected at day 35.
Pathogenicity of Avian Influenza H5N2 Viruses
Isolated in Taiwan, 2003~2012
Year
AIV
HA0
cleavage
2003
CH/031209
-REKR-
2008
2012
2012
IVPI
Pathogenicity
IVPI=0.0
LPAI
-RKKR-
IVPI=0.89
LPAI
(Potentially
Pathogenic)
CH1/120101 –RKKR-
IVPI=2.01
HPAI
GS/A703
TN/A1997
-RRKR-
IVPI=2.53
HPAI
2012
CH2/120205 –RKKR-
IVPI=2.28
HPAI
2012
CH3/120302 -RRKR-
IVPI=2.78
HPAI
2012
PT/120305
-RRKR-
IVPI=2.62
HPAI
2012
YL/120502
-RRKR-
IVPI=2.91
HPAI
Full HA and NA sequences need to be analyzed.
Courtesy of Taiwan AHRI
Production of Pandemic Influenza Vaccines
in NHRI, Taiwan
Microcarrier-based Bioreactor
2.2 L
7.5 L
30 L
150 L
200 L
(SUB)
TideCell Bioreactor (Collaboration with Medigen Inc.)
200 L
(SUB)
BioNOC
Thank you for attention
24