1
Antigenic and genetic characteristics of H5N1 viruses and candidate
H5N1 vaccine viruses developed for potential use as pre-pandemic
vaccines
The development of representative pre-pandemic H5N1 candidate vaccine viruses by
the WHO Global Influenza Programme
1
is being conducted as one step in an overall
strategy for pandemic preparedness. This summary presents the current status of the
development of new candidate H5N1 vaccine viruses and is intended to provide
guidance for national authorities on the production of pre-pandemic vaccine.
The H5N1 viruses chosen for development of pre-pandemic candidate vaccine viruses
are representative of antigenically and genetically distinct groups of viruses that have
infected humans primarily through contact with ill or dead H5N1-infected birds.
These representative candidate H5N1 vaccine viruses have been prepared by reverse
genetics and safety tested prior to release for production of pilot vaccine lots that may
be used for experimental studies and for stockpiling by governments in advance of a
possible H5N1 pandemic, should such a national policy exist. Companies are
recommended to consult individual national authorities on the H5N1 strains to be
used. Decisions should be based on the epidemiology of the circulating H5N1 viruses
that are described below.
Comparison of the previously developed (clade 1 rg A/Vietnam/1194/2004 and rg
A/Vietnam 1203/2004)
2
and new candidate H5N1 vaccine viruses and studies of
cross-reactivity of these pre-pandemic vaccine viruses and their relationship to newly
emerging H5N1 viruses are ongoing, and will be reported periodically by WHO.
Genetic characteristics of recent H5N1 viruses
The haemagglutinin (HA) sequences of the majority of H5N1 viruses circulating in
avian species during the past 3 years separated into two distinct phylogenetic clades
(genetic groups).
3
Clade 1 viruses circulating in Cambodia, Thailand and Viet Nam
were responsible for human infections in those countries during 2004 and 2005. Clade
2 viruses circulated in birds in China and Indonesia during 2003–2004 and
subsequently during 2005–2006 spread westwards to the Middle East, Europe and
Africa. This latter genetic group of viruses has been principally responsible for human
infections during the later part of 2005 and 2006. Six sub-clades of clade 2 have been
distinguished, three of which (subclades 1, 2 and 3) also differ in geographical
distribution and have been largely responsible for human cases in Indonesia, in
countries in the Middle East, Europe and Africa, and in China, respectively (
Fig. 1
).
Antigenic characteristics of recent H5N1 viruses
The antigenic relationships between the HAs of human isolates representative of clade
1 and three subclades of clade 2 were compared by haemagglutination inhibition (HI)
1
See http://www.who.int/csr/disease/influenza/mission/en/
2
See
http://www.who.int/csr/disease/avian_influenza/guidelines/avian_influenza_prototype_strains/en/index
.html
3
See http://www.cdc.gov/ncidod/EID/vol11no10/05-0644.htm
2
tests using post-infection ferret antisera. Reciprocal cross-reactions in HI tests
demonstrated antigenic similarity of HAs within the same genetic clade and
distinguished representatives of different clades (
Table 1
), with the exception of
viruses from the Karo cluster
4
represented by A/Indonesia/CDC625/2006. Viruses
from this family cluster were antigenically distinguishable from the majority of
human isolates represented by A/Indonesia/5/2005 and A/Indonesia/CDC357/2006
(subclade 1), and appeared antigenically more closely related to H5N1 viruses in
subclade 2.
New candidate vaccine viruses
Viruses representative of subclade 1 (A/Indonesia/5/2005) and subclade 2 (A/Bar
headed goose/Qinghai/1A/2005, A/Whooper swan/Mongolia/244/2005 and
A/turkey/Turkey/1/2005) were selected
5
for the preparation of reverse genetics
modified reassortant vaccine viruses using the laboratory reference strain A/PR8/34 as
donor of the polymerase, nucleoprotein, matrix and non-structural protein genes. HI
analysis confirmed that the reassortant candidate vaccine viruses were antigenically
similar to the parent viruses and the majority of recent isolates within the same clade.
On the basis of more recent data, a subclade 3 vaccine virus is also being prepared
from A/Anhui/1/2005.
Recommended use of candidate pre-pandemic H5N1 vaccine viruses
Pre-pandemic vaccines have been produced by manufacturers using clade 1 viruses
(rg A/Vietnam/1194/2004 (NIBRG-14) and rg A/Vietman/1203/2004 (CDCRG-1 and
SJRG-161052)). Clinical trials have been conducted or are under way in several
countries and stockpiling of clade 1 vaccines has begun in some countries. Because it
is not known if the next influenza pandemic will be caused by H5N1 viruses or which
of the clades or subclades of H5N1 would be responsible, should one occur, clinical
trials using clade 1 viruses should continue as an essential element in pandemic
preparedness to maximize data available on priming, cross-reactivity and cross-
protection by vaccine viruses from different clades and subclades.
On the basis of the geographical spread, the epidemiology, and the antigenic and
genetic properties of the H5N1 viruses isolated from humans during the past 12
months, national authorities may recommend the use of one or more of the following
H5N1 candidate vaccine viruses for pilot lot vaccine production and subsequent
stockpiling of vaccines, should relevant national policies exist:
An A/Indonesia/5/2005-like virus
An A/Bar headed goose/Qinghai/1A/2005-like virus
6
An A/Anhui/1/2005-like virus
7
4
See http://www.who.int/csr/don/2006_05_31/en/index.html
5
See http://www.who.int/csr/disease/avian_influenza/guidelinestopics/en/index5.html
6
Candidate vaccine viruses also include A/turkey/Turkey/1/05 and A/Whooper
swan/Mongolia/244/2005.
7
Candidate vaccine virus in preparation.
3
Additional pre-pandemic vaccine candidates will be developed as H5N1 viruses
continue to evolve, and will be announced as they become available.
Institutions, companies and others interested in pandemic vaccine development who
wish to receive these prototype strains should contact the WHO Global Influenza
Programme at whoinfluenza@who.int or the institutions listed in the publications at
the above web site.
Fig. 1
Evolution of the H5N1 haemagglutinin gene
Hong Kong/156/97
Vietnam/JP14/05
ck/Cambodia/013LC1b/05
Vietnam/1194/04 *
Vietnam/1203/04 *
Vietnam/HN30408/05
Thailand/16/04
Vietnam/JPHN30321/05
Clade 1
Hong Kong/213/03 *
Indonesia/CDC523/06
Indonesia/CDC699/06
Indonesia/CDC326/06
Indonesia/5/05*
Indonesia/CDC184/05
Indonesia/7/05
dk/KulonProgoBBVET9/04
ck/Indonesia/CDC25/05
Indonesia/6/05
ck/Brebes/BBVET2/05
Indonesia/CDC594/06
#
ck/Dairi/BPPVI/05
Clade 2
Subclade 1
ck/Yunnan/374/04
ck/Yunnan/115/04
ck/Yunnan/493/05
ck/Yunnan/447/05
dk/Guangxi/13/04
ck/Guangxi/12/04
whooping swan/Mongolia/244/05 *
bar headed gs/Qinghai/1A/05 *
Turkey/65596/06
Turkey/15/06
Iraq/207NAMRU3/06
ck/Nigeria/641/06
mld/Italy/332/06
turkey/Turkey/1/05 *
Egypt/2782NAMRU3/06
Djibouti/5691NAMRU3/06
ck/Nigeria42/06
migratory dk/Jiangxi/2136/05
gs/Kazakhstan/464/05
ck/Krasnodar/01/06
Azerbaijan/011162/06
swan/Iran/754/06
Clade 2
Subclade 2
dk/Laos3295/06
Anhui/1/05 *
Anhui/2/05
Japanese white-eye/Hong Kong/1038/06
ck/Malaysia935/06
Vietnam/30850/05
Guangxi/1/05
dk/Hunan/15/04
qa/Guangxi/575/05
dk/Vietnam/Ncvdcdc95/05
Clade 2
Subclade 3
migratory dk/Jiangxi/1653/05
gs/Guangdong/1/96
5 nucleotides
Indonesia/CDC625/06
#
* Candidate vaccine reference viruses
#
Karo family cluster
Table 1. HEMAGGLUTINATION INHIBITION REACTIONS OF INFLUENZA H5N1 VIRUSES
REFERENCE FERRET ANTISERA
STRAIN DESIGNATION
VN/1194 VN/1203
TH/16
IND/5-R
IND/357 IND/625*
TK/15
WS/244-R
BHG/1A-R TY/TK/1-R
DK/HU/15
ANH/1
GI/1
1 A/VIETNAM/1194/2004
a
640
160
nd
20
nd
nd
nd
20
20
<20
nd
nd
nd
2 A/VIETNAM/1203/2004
a
320
160
160
10
<10
80
<10
<10
40
<20
160
10
20
3 A/THAILAND/16/2004
a
nd
160
160
10
<10
40
<10
<10
40
nd
80
<10
10
5 A/INDONESIA/5/2005 CDC RG-2
b
80
<10
<10
320
320
160
40
20
80
40
40
40
20
6 A/INDONESIA/CDC357/2006
b
nd
40
20
320
640
80
40
40
80
nd
20
20
10
7 A/INDONESIA/CDC625/2006
b
*
nd
40
10
80
40
1280
40
40
160
nd
40
40
20
8 A/TURKEY/15/2005
c
80
20
<10
40
40
1280
640
640
1280
320
20
40
40
9 A/W. SWAN/MG/244/2005 SJRG-163243
c
80
20
10
40
80
640
320
320
640
320
20
10
10
10 A/B-H GOOSE/QINGHAI/1A/2005 SJRG-163222
c
80
10
<10
40
80
320
80
1280
320
160
40
20
20
11 A/TURKEY/TURKEY/1/2005 NIBRG-23
c
80
<40
nd
80
nd
nd
nd
320
160
320
nd
nd
nd
12 A/DUCK/HUNANWG/15/2004
d
nd
80
80
20
20
20
<10
<10
20
nd
160
160
nd
13 A/ANHUI/1/2005
d
nd
40
20
<10
<10
20
10
10
40
nd
160
640
160
14 A/GUANGXI/1/2005
d
nd
10
20
20
10
10
<10
<10
40
nd
nd
320
160
a
Clade 1
b
Clade 2, Subclade 1
c
Clade 2, Subclade 2
d
Clade 2, Subclade 3
* Karo family cluster - father of 10 y.o. M