﻿JOURNAL OF VIROLOGY, Dec. 2007, p. 12911­12917 Vol. 81, No. 23
0022-538X/07/$08.000 doi:10.1128/JVI.01522-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Epitope Mapping of the Hemagglutinin Molecule of a Highly
Pathogenic H5N1 Influenza Virus by Using
Monoclonal Antibodies
Nikolai V. Kaverin,1
Irina A. Rudneva,1
Elena A. Govorkova,1,2
Tatyana A. Timofeeva,1
Aleksandr A. Shilov,1
Konstantin S. Kochergin-Nikitsky,1
Piotr S. Krylov,1
and Robert G. Webster2,3
*
D. I. Ivanovsky Institute of Virology, 123098 Moscow, Russia,1
and Department of Infectious Diseases,
St. Jude Children's Research Hospital,2
and Department of Pathology, University of Tennessee,3
Memphis, Tennessee 38105
Received 11 July 2007/Accepted 9 September 2007
We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the
three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Viet-
nam/1203/04 (H5N1) HA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1)
virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to
A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants'
HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the
complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted
with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior:
each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino
acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not
previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1)
virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized
(N. V. Kaverin et al., J. Gen. Virol. 83:2497­2505, 2002). The hemagglutination inhibition reactions of the
MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes
but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the
recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA
antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for character-
izing pathogenesis and vaccine design.
Since 1997, highly pathogenic avian H5N1 influenza viruses
have caused serious outbreaks in poultry farms and markets
and have caused infection in humans with 50% mortality rate
(30). Between 2003 and 2007 H5N1 influenza viruses spread
rapidly through Southeast Asia and emerged in Western China
(3), Africa (9), Turkey (15), and Siberia (18). The rapid dis-
semination and ongoing evolution of avian H5N1 viruses, the
possibility of future interhuman transmissibility, and the ab-
sence of anti-H5 herd immunity in humans raise concern about
the pandemic potential of these viruses (13, 28) and lend new
urgency to elucidation of the structure and evolution of their
proteins.
The viral hemagglutinin (HA) surface glycoprotein is the
primary target of neutralizing antibodies. However, few of
the 16 HA subtypes have been characterized with respect to
the localization and structure of their antigenic sites on the
three-dimensional structure of the HA molecule. For many
years the three-dimensional structure of HA was available only
for the H3 subtype (29). The H3 structure was used to map
antigenic sites on the H1 (1) and H2 (27) HA molecules and to
begin characterizing the antigenic structure of the H5 HA
molecule (17). After the X-ray crystallographic structures of
H5 and H9 HA were reported (6, 7), the H5 HA molecule was
antigenically mapped in greater detail (10), and mapping of H9
HA was initiated (11). The localization and fine structure of
two H5 antigenic sites have been described (10). Site 1 is an
exposed loop comprising HA1 residues 140 to 145 (H3 num-
bering here and throughout the text) that corresponds to an-
tigenic sites A of H3 (29) and Ca2 of H1 (1), and site 2
comprises two subsites, one (HA1 residues 156 and 157) that
corresponds to site B in the H3 subtype (29) and one (HA1
residues 129 to 133) that corresponds to site Sa in the H1
subtype (1).
The recently defined three-dimensional HA structure of the
highly pathogenic H5N1 strain A/Vietnam/1203/04 (24) differs
from that of A/Duck/Singapore/3/97 (H5N3) virus (6, 7) and
bears some similarity to the HA of the H1N1 human 1918
pandemic virus. Because of the continuous evolution of H5N1
viruses (4, 13, 20, 23, 28) and the emergence of new antigenic
variants (8, 24), high-yield reassortant strains must be con-
stantly redefined for vaccine production (5, 14, 25, 26), and
immunologic diagnostic tests must be frequently updated (2).
We therefore antigenically mapped the HA molecule of the
A/Vietnam/1203/04 virus. We also characterized some mono-
* Corresponding author. Mailing address: Department of Infectious
Diseases, St. Jude Children's Research Hospital, 332 North Lauder-
dale St., Memphis, TN 38105-2794. Phone: (901) 495-3400. Fax: (901)
523-2622. E-mail: robert.webster@stjude.org.

Published ahead of print on 19 September 2007.
12911
clonal antibodies (MAbs) of the panel used to study the cur-
rently circulating highly pathogenic H5N1 influenza virus
strains (5, 8, 23). This information about the epitopes recog-
nized by these MAbs will expand their usefulness in studies of
new H5N1 isolates.
The HA amino acid sequence of the emerging H5N1 influ-
enza viruses is being monitored, but the antigenic HA epitopes
of these viruses have never been mapped on their three-di-
mensional HA structures by generating and characterizing es-
cape mutants. Our mapping revealed that the HA antigenic
structure of recent H5N1 isolates differs substantially from that
of a low-pathogenicity H5 strain described earlier (10) and is
rapidly evolving.
MATERIALS AND METHODS
Viruses. A reverse genetics-derived influenza virus containing the HA and
neuraminidase (NA) genes of A/Vietnam/1203/04 (H5N1) virus in the genetic
background of the high-growth master strain A/Puerto Rico/8/34 (H1N1)
(VNH5N1-PR8/CDC-RG) was kindly provided by R. Donis (Centers for Disease
Control and Prevention, Atlanta, GA). The HA gene of this virus had been
modified by site-specific mutagenesis to delete the multibasic amino acids at the
HA cleavage site (H5-PR8). This virus is not pathogenic to chickens or ferrets
and can be used in minimal-biosafety level laboratories. All other H5N1 viruses
were obtained from the collaborating laboratories. The mouse-adapted variant
of A/Mallard/Pennsylvania/10218/84 (H5N2) virus and its escape mutants have
been described (10, 22). Viruses were propagated by growth for 48 h in the
allantoic cavities of 10-day-old embryonated chicken eggs at 37°C and were
stored at 80°C until used.
MAbs. To select and characterize escape mutants, we used a panel of seven MAbs
to the HA of A/Vietnam/1203/04 (H5N1) influenza virus (VN04-2, VN04-8,
VN04-9, VN04-10, VN04-13, VN04-15, and VN04-16) and MAb 777/1 to the HA of
A/Chicken/Pennsylvania/8125/83 (H5N2) virus. The MAbs were prepared by a mod-
ification of the method described by Kohler and Milstein (12) as previously de-
scribed (8). Briefly, female 8-week-old BALB/c mice (Jackson Laboratories, Bar
Harbor, ME) were anesthetized with isoflurane and intranasally inoculated with 50
l of 104
50% egg infective dose/mouse of recombinant virus generated at St. Jude
Children's Research Hospital and carrying HA (modified multibasic amino acid at
the cleavage site) and NA genes from A/Vietnam/1203/04 (H5N1) virus and internal
genes from A/Puerto Rico/8/34 (H1N1) virus in phosphate-buffered saline. Six weeks
later mice were intraperitoneally boosted with 15 g of HA of concentrated, and
purified virus mixed 1:1 with incomplete Freund adjuvant (Sigma-Aldrich, Inc., St.
Louis, MO). Three weeks later mice were intraperitoneally boosted with the same
immunogen composition, followed by intravenous injection of 10 g of HA of
concentrated and purified virus 3 weeks later.
Selection of escape mutants. As described previously (10), virus was incubated
with an excess of MAb for 1 h at 20°C, and the mixture was inoculated into
10-day-old embryonated chicken eggs and incubated for 48 h at 37°C. Virus was
harvested and used for limiting-dilution cloning in embryonated chicken eggs.
HI test. Hemagglutination inhibition (HI) testing was performed with 0.5%
chicken red blood cells by a standard method (16).
PCR amplification and sequencing. Viral RNA was isolated from allantoic
fluid by using the RNeasy minikit (QIAGEN) as specified by the manufacturer.
Reverse transcription and subsequent PCR was performed with primers specific
for the HA gene segment (primer sequences are available upon request). PCR
products were purified with a QIAquick PCR purification kit (QIAGEN). The
DNA template was sequenced by using a DNA ABI Prism 3130 sequencer
(Applied Biosystems) and BigDye Terminator v3.1 kit; DNA sequences
were completed and edited by using DNASTAR sequence analysis software
(DNASTAR, Inc.).
Nucleotide sequence accession numbers. The nucleotide sequences obtained
in the present study have been deposited in the GenBank database (accession
numbers EU122394 to EU122406).
RESULTS
Selection and antigenic characterization of escape mutants.
The recombinant virus containing the HA and NA genes of
A/Vietnam/1203/04 (H5N1) virus (VNH5N1-PR8/CDC-RG) was
used as the wild-type virus for the selection of escape mutants.
The MAbs used for selection were generated against the HA
glycoprotein of A/Vietnam/1203/04 virus (seven MAbs) or
A/Chicken/Pennsylvania/8125/83 virus (one MAb 777/1). Thir-
teen mutants were selected: two mutants with each of the MAbs
VN04-2, VN04-9, VN04-13, VN04-15, and 777/1 and one mutant
with each of the MAbs VN04-8, VN04-10, and VN04-16. The
mutants were selected in three separate experiments: in the first
experiment three MAbs were used and six mutants were selected,
in the second experiment four MAbs were used and six mutants
were selected, and one mutant was selected in the third experi-
ment. The mutants are designated by the MAb number and (in
parentheses) the clone number: for example, the mutants m2(1)
and m2(4) were selected by MAb VN04-2.
All selected escape mutants were tested by HI assay with the
panel of MAbs (Table 1). The results allowed us to operation-
ally define two epitopes: one reacting with MAbs VN04-9,
VN04-10, and VN04-16 (group 1) and one reacting with MAbs
TABLE 1. Effect of amino acid changes in the HA of A/Vietnam/1203/04 (H5N1) virus on the antigenic specificity of escape mutants
Escape mutant
HA amino acid changea
Reactivity with MAbb
:
H3 numbering H5 numbering VN04-2 VN04-8 VN04-9 VN04-10 VN04-13 VN04-15 VN04-16 777/1
m2(1) S126Y, I155T S121Y, I151T  0 0 0 0 0 0 0
m2(4) R166G R162G  0 0 0 0 0 0 0
m8(9) K144E K140E 0  0 0 0  0 2
m9(5) K156E K152E 0 0   0 0  
m9(6) T160A, K193E T156A, K189E 0 0  0  0 0 
m10 K156E K152E 0 0   0 0  
m13(13) S145F S141F 0  0 0   0 
m13(16) D187N, K193E D183N, K189E 0 0  0  1 0 
m15(17) S145P S141P 0  0 0 2  0 
m15(20) G143E G139E 0  0 0 0  0 0
m16 K156N K152N 0 0   0 0  
m777/1(2) S145P S141P 0  0 0 2  0 
m777/1(4) S145T S141T 0  0 0 0 2 0 
a
Amino acid numbering is based on H3 HA and H5 HA (6).
b
Values are the differences in log2 units between the HI titers of MAbs in reactions with the wild-type VNH5N1-PR8/CDC-RG (H5N1) virus and with its escape
mutants. 0, The HI titer of the MAb does not differ from the titer with the wild-type virus. , The HI titer at least 32-fold (5 log2) less than the titer with the wild-type
virus.
12912 KAVERIN ET AL. J. VIROL.
VN04-8 and VN04-15 (group 2). In contrast, the mutants se-
lected with VN04-2 were resistant solely to VN04-02, which,
therefore, could not be assigned to any epitope on the basis of
the HI test. The mutants selected with MAbs VN04-13 and
777/1 (group 3) exhibited a complex pattern of specificity that
overlapped both epitopes defined by group 1 and group 2
MAbs. Two mutants selected with VN04-13 differed in their
reactions. Whereas mutant m13(13) failed to react with
VN04-8 and VN04-15, mutant m13(16) reacted with both, but
not with MAb VN04-9. Of the mutants selected by MAbs to
A/Vietnam/1203/04 virus, only m2(1), m2(4), and m15(20) re-
acted with MAb 777/1 (Table 1).
The data demonstrated that the reactions of MAbs VN04-9,
VN04-10, and VN04-16 overlapped, suggesting that these
MAbs reacted with the same antigenic site, whereas MAbs
VN04-8 and VN04-15 reacted with a different antigenic deter-
minant. MAb VN04-2 had an extremely narrow specificity and
therefore could not be assigned to either epitope group on the
basis of HI data alone. MAbs VN04-13 and 777/1 could not be
assigned to either group for the opposite reason: they failed to
react with mutants selected by the MAbs belonging to either
group. This result suggested that they had unusually broad
epitope specificity.
Sequence analysis of the escape mutants. To identify the
specific amino acids that reacted with the MAbs, we sequenced
the HA gene. We found that the escape mutants carried either
one or two amino acid changes in the HA1 subunit but none in
the HA2 subunit.
The mutations selected by group 1 MAbs (VN04-9, VN04-
10, and VN04-16) were located between positions 155 and 166,
corresponding to a part of antigenic site B in H3 HA and to the
site Sa in H1 HA. MAb VN04-2 also recognized this site,
because it selected mutants carrying amino acid changes at
positions 126, 155, and 166. The amino acid substitutions se-
lected by group 2 MAbs (VN04-8 and VN04-15) were located
in the 140-145 loop, that is, in the site corresponding to anti-
genic site A in H3 HA (Table 1).
Two MAbs selected escape mutants with unusual patterns of
amino acid changes. MAb VN04-13 selected two mutants with
amino acid changes in two widely separated areas. The mutant
m13(13) carried the substitution S145F in the loop, which
explained its inability to react with MAbs VN04-8 and VN04-
15. The other mutant, m13(16), had no substitutions in this site
but carried two mutations (D187N and K193E) in a region
corresponding to a part of site B in H3 HA. The substitution
K193E was clearly antigenically significant, because MAb
VN04-13 failed to react with the mutant m9(6), which also
carried this substitution. Further, Philpott et al. (17) have re-
ported an amino acid change at position 193 in an H5 escape
mutant.
MAb 777/1 also recognized amino acid substitutions in two
distantly located antigenic sites: the region corresponding to
site B in H3 HA (positions 156 and 193) and position 145 in the
loop. However, both mutants selected with MAb 777/1 had a
substitution at position 145: S145P in mutant m777/1(2) and
S145T in mutant m777/1(4).
Clearly, epitopes reacting with MAbs VN04-13 and 777/1
overlap two antigenic sites, one corresponding to site A and
one corresponding to site B in H3 HA. The other MAbs ex-
hibited narrower specificity, selecting escape mutants with
amino acid changes in sites corresponding to either antigenic
site A in H3 (MAbs VN04-8 and VN04-15) or site B in H3
and site Sa in H1 (MAbs VN04-2, VN04-9, VN04-10, and
VN04-16).
Each of three escape mutants--m2(1), m9(6), and m13(16)--
that were selected with three different MAbs were double mu-
tants carrying two HA amino acid changes. The HI data did not
allow us to ascribe any antigenic effect to the substitutions T160A
and D187N or to discern whether the antigenic change of mutant
m2(1) was caused by substitution S126Y or I155T. However, all of
these substitutions are located in antigenic areas, and their effect
on antigenic specificity cannot be excluded.
HI testing of the anti-A/Vietnam/1203/04 MAbs with escape
mutants of A/Mallard/Pennsylvania/10218/84 virus. All of the
MAbs except VN04-8 reacted with the A/Mallard/Pennsylva-
nia/10218/84 (H5N2) virus, allowing us to use single, double,
and triple escape mutants of this virus generated and charac-
terized in our previous study (10) to further examine the spec-
ificity of the MAbs. In HI testing MAb VN04-2 failed to react
with mutants having the amino acid substitution N129D (Table
2). The reactions of MAbs VN04-9, VN04-10, and VN04-16
were affected by amino acid changes at position 156, which was
expected on the basis of the data of the sequencing of the
escape mutants selected with these MAbs (Table 1). However,
MAb VN04-10 lost the ability to react with mutants carrying
the K156N substitution and yet retained reactivity with those
TABLE 2. Reactions of MAbs with escape mutants of A/Mallard/Pennsylvania/10218/84 (H5N2) virus in HI assay
Escape mutant
HA amino acid changea
Reactivity with MAbb
:
H3 numbering H5 numbering VN04-2 VN04-9 VN04-10 VN04-13 VN04-15 VN04-16 777/1
m46(7) N129D N124D  0 0 0 0 0 0
m46(8) K157M K153M 0 0 0 0 2 0 0
m55(2) K156N K152M 0   0 0  4
m58(1) D131N D126N 0 2  0   1
m24B9 R144G R140G 0 0 0 0  0 0
m46(7)-55 N129D, K156T N124D, K152T   0 0 2  0
m46(7)-55-24B9 N129M, K156T, P140L N124D, K152T, P136L   2    2
m55(2)-24B9 K156N, N142K K152T, N138K 0   0   
m58(1)-24B9 D131N, S145P D126N, S141P 0 2     
a
See Table 1, footnote a.
b
Designations are the same as in Table 1. Values are the differences in log2 units between the HI titers of MAbs in the reactions with the wild-type A/Mallard/
Pennsylvania/10218/84 (H5N2) virus and with its escape mutants.
VOL. 81, 2007 ANTIGENIC EPITOPES OF INFLUENZA H5N1 VIRUS HA 12913
carrying K156T. Reactions of VN04-15 were affected not only
by changes in the loop corresponding to site A in H3 HA
(P140L, N142K, and R144G) but also by the change D131N,
located in the site corresponding to site B in the H3 HA, which
generates a new glycosylation site (10). Reaction of the escape
mutants with MAb 777/1 was abolished by the amino acid
substitution S145P and reduced by the substitution K156N,
confirming the ability of this MAb to react with epitopes in two
different antigenic sites.
The results obtained with escape mutants of both A/Viet-
nam/1203/04 (H5N1) and A/Mallard/Pennsylvania/10218/84
(H5N2) viruses allowed us to divide the MAbs into three
groups on the basis of their epitope recognition. Group 1
MAbs (VN04-2, VN04-9, VN04-10, and VN04-16) react with
the complex epitope comprising parts of antigenic site B of the
H3 HA and site Sa of H1 HA. Group 2 MAbs (VN04-8 and
VN04-15) react with the epitope corresponding to antigenic
site A in H3. However, MAb VN04-15 is also sensitive to the
acquisition of the glycosylation site at position 131. Group 3
MAbs (VN04-13 and 777/1) recognize amino acid changes in
two distantly located antigenic sites. Figure 1 shows a sche-
matic representation of the epitopes of the MAbs overlapping
two antigenic sites.
Cross-reactions of the anti-A/Vietnam/1203/04 MAbs with
highly pathogenic H5N1 viruses. In HI testing, both the H5N1
viruses preceding A/Vietnam/1203/04 virus and those isolated
later were resistant to some MAbs in the panel (Table 3).
These strains exhibited differences from one another and from
A/Vietnam/1203/04 (H5N1) virus in their reactions with the
MAbs. The reaction was either slightly enhanced compared to
the reaction with A/Vietnam/1203/04 (H5N1) virus, or de-
creased, or completely abolished. The differences did not co-
incide with clade and subclade division based on phylogenetic
analysis of the H5 HA gene. We compared the amino acid
sequences in the HA1 polypeptide chain region that showed
amino acid changes in the escape mutants (positions 125 to
193) (Table 4). In several cases the amino acid changes were
positioned identically in the nonreactive strains and in the
escape mutants of A/Vietnam/1203/04 and A/Mallard/Pennsyl-
vania/10218/84 viruses. The nonreactivity or reduced reactivity
of the strains A/Chicken/Jogiakarta/BBVet-IX/04 and A/Duck/
Laos/3295/06 with MAbs VN04-8 and VN04-15 (Table 3) may
be explained by nonconservative amino acid differences be-
tween A/Vietnam/1203/04 virus and these strains in the region
of the loop (Table 4). The failure of A/Duck/Laos/3295/06 to
react with the MAb VN04-13 can be ascribed to the substitu-
tion S145F, which was observed in the mutants m15(17) and
m58(1)-24B9 that did not react with this MAb. The strains that
were nonreactive with MAb VN04-2 (Table 3) had an aspartic
acid residue at position 129 (Table 4), as did the mutants
FIG. 1. Schematic representation of the antigenic sites and the
epitopes that react with MAbs VN04-13 (A) and 777/1 (B) on the
globular head of the HA H5 HA molecule. Images were created with
RasMol 2.6, and the HA structure was obtained from the Protein Data
Bank (PDB accession number 1JSM). Amino acid positions are des-
ignated in H3 numbering.
TABLE 3. Cross-reactivity of anti-A/Vietnam/1203/04 (H5N1) HA MAbs with H5N1 viruses
HA phylogeny Virus
Reactivity with MAba
:
VN04-2 VN04-8 VN04-9 VN04-10 VN04-13 VN04-15 VN04-16
Clade 1 A/Vietnam/1194/04 1 0 0 0 0 1 0
A/Hong Kong/213/03 0 1  0 2 1 1
Clade 2A, subclade 1 A/Chicken/Indonesia/PA/03 3 1  0 2 0 0
A/Chicken/Malang/BBVet-IV/04 1 1  0 2 0 0
A/Chicken/Jogjakarta/BBVet-IX/04    0 1  2
Clade 2E, subclade 2 A/Whooper swan/Mongolia/244/05  0  0 2 0 2
Clade 2F, subclade 3 A/Duck/Laos/3295/06   3 0   3
a
Designations are the same as in Table 1. Values are the differences in log2 units between the HI titers of MAbs in the reactions with the A/Vietnam/1203/04 virus
and with the other H5N1 viruses.
12914 KAVERIN ET AL. J. VIROL.
m46(7) and m46(7)-55, which were nonreactive with this MAb
(Table 2). The strains with amino acid changes at both position
160 and position 193 failed to react with MAb VN04-9, as had
the escape mutants with amino acid substitutions at these po-
sitions. Although we could not identify any specific antigenic
alteration caused by amino acid substitution T160A in mutant
m9(6), it is noteworthy that this amino acid change is a reverse
mutation: the change A160T occurred in several H5N1 strains
after 2003 (4, 24). Obviously, amino acid changes in the anti-
genic sites revealed by the analysis of escape mutants are
involved in the evolution of the H5 HA. It is worth noting that
the distribution of antigenically significant amino acid changes
in the HA of the H5N1 strains was not related to HA-based
clades and subclades (Tables 3 and 4).
DISCUSSION
We have described the first characterization of the antigenic
structure of the HA of a highly pathogenic H5N1 virus through
the selection and study of escape mutants. The evolution of
highly pathogenic H5N1 strains has to date been monitored by
sequencing the genomes of isolates and using polyclonal sera
and MAbs to detect antigenic changes. Our results will refine
the detection of antigenically important changes in the evolv-
ing H5 HA.
The location and structure of influenza virus HA antigenic
epitopes was first characterized in 1981 in the three-dimen-
sional model of the H3 subtype (29). Four antigenic sites were
described (A, B, C, and D), and a fifth (E) was later demon-
strated (21). The antigenic sites of H1 and H2 subtype HAs
were then characterized and mapped on the H3 HA three-
dimensional structure (1, 27), and a preliminary map of the H5
HA antigenic sites was also based on the X-ray model of H3
HA (17). The locations of five H5 HA antigenic epitopes were
identified, but their size and fine structure was not described,
because only six amino acid changes were observed. After the
X-ray crystallographic structures of H5 and H9 molecules were
reported (6, 7), we mapped the antigenic sites on their surfaces
by generating and analyzing escape mutants (10, 11). Amino
acid changes in the escape mutants of the low-pathogenicity
avian A/Mallard/Pennsylvania/10218/84 (H5N2) virus were
grouped in two areas in the HA molecule: one corresponding
to antigenic site A in H3 HA (a loop at the side of HA
molecule) and another corresponding to a part of antigenic site
B in the H3 HA molecule and partially overlapping the anti-
genic site Sa in H1 HA. The recent extensive spread of highly
pathogenic influenza H5N1 viruses since 2003 prompted us to
characterize the antigenic structure of the H5 HA using the
human isolate A/Vietnam/1203/04 (H5N1), which represents
clade 1 on the H5 HA phylogenetic tree.
We identified several important differences between a recent
H5N1 strain and the previously described H5 HA molecules
(10, 17). First, the positions of amino acid changes in the
escape mutants of the A/Vietnam/1203/04 (H5N1) virus dif-
fered from those we previously described in A/Mallard/Penn-
sylvania/10218/84 (H5N2) mutants (10), with the exception of
positions 144, 145, and 156, although they are located within
the same areas on the surface of the HA molecule. Second,
when the positions coincide, the amino acid changes are dif-
ferent (e.g., K156E and K156N). All of these observations are
likely to reflect a difference in HA conformation between the
more recent H5N1 strain and A/Mallard/Pennsylvania/
10218/84 (H5N2) virus. The amino acid changes in positions
previously unreported in escape mutants (126, 143, 155, 160,
166) that altered the reactivity with the MAbs were located
both in the loop and in the part corresponding to antigenic site
B of H3 HA. Interestingly, in the present study and in our
previous study (10), all antigenically significant amino acid
substitutions were located exclusively in areas corresponding to
antigenic sites A and B of H3 HA and the antigenic site Sa of
H1 HA. Not a single MAb against A/Vietnam/1203/04 (H5N1),
A/Chicken/Pennsylvania/10218/83 (H5N2), or A/Chicken/
Pennsylvania/8125/83 (H5N2) viruses in our studies selected an
escape mutant with amino acid changes in the areas at the side
of the HA molecule designated as sites 2, 3, and 4 by Philpott
et al. (17). It seems plausible that the repertoire of immuno-
competent antibody-producing cells (at least in mice) is di-
rected largely against the upper surface of the H5 HA mole-
cule.
An unexpected finding was the ability of at least two MAbs
to overlap widely separate antigenic sites. Of the two mutants
selected by MAb VN04-13, one had an amino acid change
(S145F) in the loop corresponding to site A in H3, while the
TABLE 4. Amino acid changes in HA of the H5N1 viruses
HA phylogeny Virus
Amino acid at positiona
:
125 129 133 142 144 145 158 159 160 163 166 178 185 193
120 124 129 138 140 141 154 155 156 159 162 174 181 189
Clade 1 A/Vietnam/1203/04 S S L Q K S N S T T R V P K
A/Vietnam/1194/04 S S L Q K S N S T T R V P K
A/Hong Kong/213/03 N S L Q K S N N A T R V P R
Clade 2A, subclade 1 A/Chicken/Indonesia/PA/03 S D S Q K S N S A T R V P R
A/Chicken/Malang/BBVet-IV/04 S N S Q K S N S A T R V P R
A/Chicken/Jogjakarta/BBVet-IX/04 S D S L R S N S T I R V P K
Clade 2E, subclade 2 A/Whooper swan/Mongolia/244/05 S D S Q R S D N A T R V P R
Clade 2F, subclade 3 A/Duck/Laos/3295/06 S D S Q T P N N T T R I S K
a
Amino acid numbering in the top row is based on H3 HA. Amino acid numbering in the bottom row is based on H5 HA. Amino acid substitutions with respect
to the HA of A/Vietnam/1203/04 (H5N1) are indicated in boldface. Amino acid positions changed in the HA of escape mutants are indicated in italics.
VOL. 81, 2007 ANTIGENIC EPITOPES OF INFLUENZA H5N1 VIRUS HA 12915
other carried 2 mutations in a region corresponding to a part
of site B in H3. MAb 777/1 also reacted with two antigenic
sites, as demonstrated by its reactions with escape mutants of
A/Vietnam/1203/04 (H5N1) and A/Mallard/Pennsylvania/
10218/84 (H5N2) viruses. With some reservations, MAb
VN04-15 may also be regarded as sensitive to amino acid
substitutions in two antigenic sites. However, in this case the
amino acid change at position 131 was associated with the
acquisition of a new glycosylation site (10). The oligosaccha-
ride chain in this position might create steric hindrance at the
epitope recognized by the MAb VN04-15 in the region of the
loop.
Importantly, the MAbs overlapping two antigenic sites
(VN04-13 and 777/1) were not mixed preparations. Amino acid
changes that abolished recognition by these MAbs were de-
tected in several single mutants carrying a mutation in one
antigenic site, while the other site was unchanged and free to
bind MAbs: the mutants, therefore, would have been neutral-
ized by a mixture containing a MAb reactive with the un-
changed site. Further, escape mutants are unlikely to be se-
lected by a mixture of two MAbs reacting with different
antigenic sites, considering the mean frequency of the muta-
tions selected by MAbs (31).
The ability of some anti-H5 MAbs to overlap 2 distinct
antigenic sites may be hypothetically explained by the archi-
tecture of the H5 HA molecule. Sites A and B are situated far
apart in the three-dimensional structure of H3 HA molecule
(29) but are much closer in H5 HA (10, 11). It would be
premature to speculate whether this proximity may influence
the selection of antigenic variants under natural conditions.
However, a neutralizing antibody that reacts with two antigenic
sites rather than one might offer the evolving virus broader
possibilities of escape, if a mutation in either site could confer
resistance to neutralization. The ability of some anti-H5 MAbs
to overlap two different antigenic sites suggests that if H5
viruses were to become disseminated in the human population,
the generation of drift variants of H5 HA might differ from
that observed in the human H1 and H3 viruses.
The panel of MAbs we characterized is widely used to study
currently circulating highly pathogenic H5N1 influenza viruses
(5, 8, 23). These MAbs are available from the Biodefense and
Emerging Infections Research Resources Repository (http:
//www.beiresources.org), and in 2007 were included into the
CDC influenza reagent kit. By identifying the epitopes recog-
nized by these antibodies, our findings allow the use of the
MAbs to monitor the evolution of the antigenic sites in the
currently circulating H5N1 viruses. Previous data obtained by
using this panel of MAbs can be reinterpreted on the basis of
our results, and the characterization of future isolates will
acquire a new dimension, in which the antigenic changes that
are detected can be connected with amino acid substitutions at
specific antigenic sites.
The antigenic sites described here and in our earlier study
(10) are significantly involved in the evolution of H5N1 influ-
enza viruses. The sites can now be analyzed in detail. Clearly,
antigenic specificity can be modulated by a greater variety of
amino acid changes than previously suggested (10). The amino
acid substitutions in the highly pathogenic H5N1 isolates (4,
24) occur within the antigenic sites described here and in our
previous study (10), and some occur in precisely the same
positions as those in the escape mutants described here and in
our previous study (10). These coincidences suggest a certain
similarity between the evolution of the avian H5N1 viruses and
the antigenic drift of human H1 and H3 influenza virus strains.
The data presented here may be regarded as a basis for
future studies in several directions. First, it will be important to
explore whether the antigenic epitopes recognized by murine
MAbs coincide with those recognized by human antibodies.
The production of human MAbs against the H5 HA was re-
cently reported (19). Second, since the distribution of the an-
tigenically significant amino acid changes in the HA of H5N1
strains did not coincide with the clade and subclade grouping
based on phylogenetic analysis of the H5 HA genes, the choice
of a strain for vaccine preparation should take into consider-
ation not only the selection of the proper clade and subclade
but also the structure of antigenic sites of individual strains. It
will be important to establish to what extent the amino acid
substitutions in the antigenic sites affect immune protection.
Finally, last but not least, it will be of interest to find out
whether the amino acid changes in the escape mutants of the
H5N1 virus have any pleiotropic effects. In our previous studies
we revealed that escape mutations in the HA of a low-patho-
genicity mouse-adapted H5N2 and H9N2 strains may be asso-
ciated with decrease of virulence and/or the affinity to sialic
receptors (10, 11). Experiments aimed at the characterization
of these phenotypic features of the escape mutants generated
in the present study are now in progress.
ACKNOWLEDGMENTS
We thank Sharon Naron for editorial assistance. We gratefully ac-
knowledge Tien D. Nguyen from National Institute of Veterinary
Research, Hanoi, Vietnam; Malik Peiris and Yi Guan from the Joint
Influenza Research Center, Shantou University Medical College and
Hong Kong University, People's Republic of China; Bounlom
Douangngeun from National Animal Health Centre, Laos PDR; Tri
Satya Putri Naipospos from Indonesia National Committee on Avian
Flu Control and Pandemic Influenza Preparedness, Indonesia; William
B. Karesh from Wildlife Conservation Society and the Global Avian
Influenza Network for Surveillance of Wild Birds; and David Swayne
from USDA ARS for providing H5N1 influenza viruses. We thank
Ruben Donis from the Centers for Disease Control and Prevention,
Atlanta, GA, for providing genetics-derived VNH5N1-PR8/CDC-RG
influenza virus.
This study was supported by grant 07-04-00005-a from the Russian
Foundation for Basic Research; by the National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Department of
Health and Human Services, grants A195357 and A157570 and con-
tract no. HHSN266200700005C; and by the American Lebanese Syrian
Associated Charities.
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