Cell Host & Microbe
Defining Influenza A Virus Hemagglutinin
Antigenic Drift by Sequential
Monoclonal Antibody Selection
Suman R. Das,1,2,3Scott E. Hensley,1,5William L. Ince,1Christopher B. Brooke,1Anju Subba,2Mark G. Delboy,2
Gustav Russ,4James S. Gibbs,1Jack R. Bennink,1and Jonathan W. Yewdell1,*
1Laboratory of Viral Diseases, NIAID, Bethesda, MD 20892, USA
2Infectious Diseases Group, J. Craig Venter Institute, Rockville, MD 20850, USA
3Emory Vaccine Center, Emory University, Atlanta, GA 30322, USA
4Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovak Republic
5Present address: The Wistar Institute, Philadelphia, PA 19130, USA
Human influenza A virus (IAV) vaccination is limited
by ‘‘antigenic drift,’’ rapid antibody-driven escape
reflecting amino acid substitutions in the globular
domain of hemagglutinin (HA), the viral attachment
protein. To better understand drift, we used anti-
hemagglutinin monoclonal Abs (mAbs) to sequen-
tially select IAV escape mutants. Twelve selection
steps, each resulting in a single amino acid sub-
stitution in the hemagglutinin globular domain,
were required to eliminate antigenicity defined by
monoclonal or polyclonal Abs. Sequential mutants
grow robustly, showing the structural plasticity of
HA, although several hemagglutinin substitutions
required an epistatic substitution in the neuramini-
dase glycoprotein to maximize growth. Selecting
escape mutants from parental versus sequential
variants with the same mAb revealed distinct
changes in antigenicity and the mutation landscape.
sculpts future mutation space, drift can follow
many stochastic paths, undermining its unpredict-
ability and underscoring the need for drift-insensi-
basis costing the USA alone upwards of $50 billion while killing
tens of thousands (Molinari et al., 2007). Although current
vaccines lessen the burden of influenza, they are far less effec-
tive than vaccines for other similar viral pathogens. This is due
to the ability of IAV to modulate its antigenicity on a yearly basis.
This process, termed antigenic drift, reflects the accumulation of
amino acid substitutions in the globular domain of HA (Webster
et al., 1975), the principal target of Abs that neutralize IAV
HA initiates the infectious cycle by binding terminal sialic
and cellular membranes. Sequencing escape mutants of A/PR/
8/34 (H1N1) (PR8) selected by neutralizing monoclonal Abs
(mAbs) (Caton et al., 1982; Gerhard et al., 1981) revealed five
largely nonoverlapping immunodominant antigenic sites. Sa
and Sb (strain specific) are located at the tip of the globular
domain, while Ca1and Ca2and Cb (crossreactive) are located
toward the stem of H1 HA. Based largely on the correlation of
antigenic sites with the degree of variation observed in drifted
field isolates, it is believed that ‘‘drift’’ results strictly from anti-
genic escape. Recent results, however, suggest that selection
for other factors, such as HA receptor specificity and avidity,
and epistatic interactions within HA and with neuraminidase
(NA) and other IAV gene products can select for changes in the
globular region that alter antigenicity (Hensley et al., 2009,
2011; Kryazhimskiy et al., 2011).
Thus, although antigenic drift of IAV has been known for nearly
80 years (Francis et al., 1947), the relative contribution of various
selective factors is uncertain. An important but largely ignored
question is why IAV rapidly drifts while other RNA viruses (e.g.,
paramyxoviruses) with equivalent mutation rates and frequency
of mAb escape mutants do not (van Wyke Coelingh et al., 1987;
Yewdell and Gerhard, 1982). To what extent is drift due to (1)
Special features of IAV transmission in human populations or
the interaction of IAV with individual hosts? (2) Enhanced
ability of HA to accept amino acid substitutions and change
antigenicity while maintaining full functionality? (3) The ability of
IAV to buffer changes in HA function with epistatic changes in
other genes, e.g., NA, a process facilitated by the segmented
nature of the IAV genome?
Here, we address the characteristics of IAV that favor anti-
genic drift by sequentially selecting IAV escape mutants with
mAbs until escape from a large panel of neutralizing mAbs is
In Vitro Modeling of Drift by Generating Sequential
The H1 HA has five spatially distinct immunodominant antigenic
sites, but single amino acid substitutions at each site only
314 Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc.
abrogate the binding of a fraction of Abs specific for each site
clonal Abs and a large panel of mAbs induced by WT virus?
We addressed this question by sequentially selecting mutants
with a panel of mAbs (Table 1). After each selection step, we
measured antigenicity using a large panel of mAbs via radioim-
munoassay (RIA) and then repeated the process with a mAb
that demonstrated little or no alteration in affinity for the sequen-
tial variant. Loss of antigenicity was gradual and predictable
based on the relationship between the epitopes recognized by
the selecting Ab and the queried panel Ab. (Figure 1A). Twelve
selection steps were required to reduce binding at least
10-fold to all but 4 of a 182 member mAb panel (Table 1, the
remaining mAbs demonstrate weak neutralization/hemaggluti-
nation inhibition [HI] activity [Yewdell, 1981]).
The reactivity of mAbs paralleled the reactivity of mouse pAbs
in mouse serum following primary or booster immunization as
measured by HI (Table 2). Postinfection ferret antisera (from
multiple sources), the WHO/CDC standard used for gauging
antigenic drift in epidemic viruses, showed a similar decrease
to the sequential (SEQ-) variants (Table 2). Sera from guinea
pig, rabbits, and birds (chickens) all showed substantial incre-
mental decreases with the SEQ variants, suggesting similarities
in the recognition of the globular domain by Ab responses
among vertebrate species (Table 2).
To examine the ability of SEQ-12 to escape immunity in vivo,
we vaccinated B6 mice with inactivated PR8 virus, intranasally
challenged them 3 weeks later with PR8 and SEQ-12 mixed at
S1). Immunization with inactivated virus resulted in a 10-fold
reduction in viral titer (p = 0.018, Student’s t test). Comparison
of SEQ-12 and PR8 variant frequencies in immunized mice
clearly demonstrates that SEQ-12 outcompetes PR8 despite
starting at a 1:5 ratio (p < 0.0001 Fisher’s exact test), whereas
in naive mice PR8 has a slight growth advantage over SEQ-12
(p = 0.03), thus confirming an overall clear growth advantage
of SEQ-12 in vaccinated mice.
These findings demonstrate that HA undergoes gradual, step-
by-step antigenic drift when confronted with individual mAbs,
and that the panel we used is relevant for escape in mice, guinea
pigs, rabbits, chickens, and ferrets, and likely humans as well,
based on the predictive value of ferret reference sera in gauging
significant antigenic drift (Smith et al., 2004).
Sequential Variants Exhibit Epistatic Changes in NA
Related to Optimizing Receptor Avidity
Sequencing of the sequential panel revealed that each selection
step was accompanied by a single nonsynonymous mutation
encoding a residue in the predicted antigenic site for each se-
lecting mAb (Table 1, located on the HA structure in Figure 1B).
For most variants, there were no additional changes in either HA
or the seven other IAV gene segments. The lack of reversion or
addition of epistatic changes in these viruses suggests that viral
fitness was not greatly affected by the selection process.
Consistent with this finding, the frequency of mAb H36-84
escape mutants in the SEQ-11 and WT stocks were indistin-
guishable (10?6.2), demonstrating that escape was not increas-
ingly constrained by the ability of the HA to accept novel
mutations after 11 selection steps. Sequential variants all grew
to similarly high titers in eggs (see Figure S1 online).
Three of the SEQ- variants possessed a nonsynonymous
mutation in the NA gene, resulting in a G357S substitution
(H5N1 numbering-based NA crystal structure 2HTY, G339S;
(Figure 1C). This mutation appeared in SEQ-8 and persisted until
reverting in SEQ-11. Although the reversion suggests that the
mutation is not a random ‘‘hitchhiker’’ offering no selective
advantage, we examined its evolutionary value by comparing
the abilities of WT and mutant NA genes to complement WT
versus mutant HAs based on virus titer after rescue and
growth in MDCK cells (Figure S1). This revealed a clear prefer-
ence of the SEQ-8 (and possibly SEQ-9 HA, p value = 0.28,
also a slightly lower success rate for rescuing virus) for mutant
versus WT NA. Conversely, WT HA or SEQ-7 HA did not demon-
strate a significant preference for mutant versus WT NA, con-
firming that the mutant NA is selected to enhance the growth
of SEQ variants.
When assessed on a per particle basis (assessed by hemag-
glutination activity), the G357S substitution reduced NA activity
as measured using a simple fluorogenic substrate (Figure 2A).
Activity differences occurred independently of glycoprotein
organization in virions, since they persisted when virus was
disrupted by adding triton X 100. When NA activity was normal-
ized to NA protein amount in virions, no differences were
observed between WT and mutant, indicating that the mutation
does not reduce NA activity (Figure 2B). Instead, by analyzing
virion protein content, we found that the G357S substitution
markedly reduces the amount of NA incorporated into virus
particles relative to HA (Figure 2C).
The reduction in per particle NA activity in SEQ variants was
associated with increased HA receptor avidity, measured by
resistance of viral HA activity to removal of terminal sialic
startingfrom initiating Met)
Table 1. Selection of Sequential Variants
Variant Antibody UsedEpitope
SEQ-1 H2-6C4B Sb
SEQ-2 H2-4B3C Sa
SEQ-12H36-80 & H36-84Ca2
Table shows antibodies used to generate the sequential variants, their
single-letter epitope designation, and location among the five antigenic
sites in the globular domain. Substitutions in SEQ-1, SEQ-3, SEQ-5,
SEQ-6, SEQ-7, SEQ-9, and SEQ-12 reiterate previously observed
substitutions in PR8 mAb escape mutants, in SEQ-2, SEQ-4, and
SEQ-8 novel amino acids in previously reported positions, and in
SEQ-10 and SEQ-11 novel residues.
Cell Host & Microbe
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Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc. 315
residues by bacterial NA (Yewdell et al., 1986). Increased avidity
first appeared in SEQ-7 and peaked in SEQ-8, where the NA
mutation was first detected (Figure 2D). E156K, appearing in
SEQ-7, also increases receptor avidity in a single mAb escape
mutant (Hensley et al., 2009), while E119K, the mutation in
SEQ-8, is in the same location as E119G, also known to increase
receptor avidity as a single substitution (Hensley et al., 2009).
This finding emphasizes the importance of optimal HA avidity
for virus growth, and the ability of epistatic changes in NA to
compensate for suboptimal HAfunction,aswerecently reported
(Das et al., 2011; Hensley et al., 2011). Substitutions in SEQ-9,
SEQ-10, and SEQ-11 all diminish HA avidity, reinforcing the
surprising conclusion that residues quite distant from the clas-
sical receptor-binding site (residue 48, altered in SEQ-11, is
located on the opposite face of the HA, more than 50 A˚from
the receptor-binding site) can influence receptor binding
sion inNA(rather thananepistatic changein anotherNAresidue)
strongly suggests that NA has little mutational latitude in
compensating for the altered function of the G357S mutation.
Notably, position 357 is highly conserved among N1 genes: in
791 isolates from 1,934 to 2,000 curated at the influenza
research database (http://www.fludb.org/), there are but 10
isolates with substitutions (G357N or G357D). Based on the
crystal structure of a related N1 NA, residue 357 is located on
the ‘‘underside’’ of the globular head ?25 A˚from the active
site (Figure 1C), likely explaining the unchanged enzymatic
activity. Rather, our evidence implicates this residue in control-
ling the efficiency of NA incorporation into virions.
Together, these data show that, as predicted by our previous
study (Hensley et al., 2009), the receptor properties of HA are
altered by immune escape-driven mutations in the classic
Figure 1. Antigenic Map of Sequential Variants
(A) The antigenicity of SEQ variants was determined by measuring the relative binding affinities of a panel of 60 mAbs via RIA. Black shows equivalent binding to
mutant and WT viruses; gray shows reduced affinity (2- to 4-fold); white shows greatly reduced affinity.
1RVX). Amino acid substitutions in escape mutants are indicated by color and label. Green shows same substitutions when selected with PR8, blue shows
same amino acid position but a new substitution, and red shows new position.
(C) Three-dimensional model of NA rendered by PyMOL software as a solid surface looking at top (top panel) and membrane proximal (bottom panel) aspects of
the tetramer molecule (using H5N1 NA crystal structure 2HTY). NA active sites (118, 151, 152, 224, 227, 276, 292, and 371) are labeled in red and substitution
G357S (H5N1 NA crystal structure 2HTY numbering) that is present in SEQ-8, SEQ-9, and SEQ-10 and decreases NA activity in green.
Also see Figure S1.
Cell Host & Microbe
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316 Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc.
antigenic sites, some of which require compensatory NA
mutations to restore fitness.
Sequential Variants Demonstrate Novel Amino Acid
All of the SEQ- substitutions are located in the sites expected for
their respective selecting mAb (Table 1 and Figure 1B). Remark-
ably, however, when compared to a panel of single selection
mutants (Caton et al., 1982; Yewdell et al., 1993), three substitu-
tions represent novel changes at a given position (depicted in
blue), while two of the substitutions are present in entirely novel
positions (depicted in red).
This suggested that the ‘‘escape space’’ of HA changes as it
accumulates amino acid substitutions. To test this hypothesis,
we used two mAbs with highly similar epitopes in the Ca site,
H17-L2 and H17-L10 (Ca8 and Ca6, respectively, in Gerhard
et al., 1981) to compare the PR8 and SEQ-7 repertoires of
mutants that escape from neutralization. H17-L10 was used to
select the unique mutation in SEQ-9 (Table 1). Both mAbs utilize
g2a heavy chains and k light chains, and require HA trimerization
to create their epitopes (Yewdell et al., 1988), consistent with
their epitopes spanning the trimer interface (Figure 3). These
two mAbs recognize PR8 and SEQ-7 with similar affinity (Figures
4D and 4E) and exhibit similar neutralization and HI potency
Analysis of large panels of escape mutants revealed the
selection of many distinct mutants (Table 3 and Table S3). The
large discrepancy in the frequency of individual mutations
(ranging from less than 1% to more than 80%) is almost certainly
due to differences in viral fitness, as such large differences
are inconsistent with relatively minor differences in codon
degeneracy of the frequencies of individual base substitutions
or with random fluctuations in mutation frequencies. Interest-
ingly, we identified mutants that acquired potential N-linked
glycosylation sites in residues that represent novel locations
for glycosylation in H1 viruses (Das et al., 2010). Two of the
residues occupy surface locations that could accommodate
an oligosaccharide (93, 172), while the third residue (210) is
located on the interior of the trimer where oligosaccharide
addition would likely interfere with trimer formation. Given the
fitness costs associated with adding novel oligosaccharides
(Das et al., 2011), it will be interesting in future studies to charac-
terize these variants biochemically and functionally.
The critical finding is that even among the more abundant
mutants, there were large or even absolute differences in their
presence in WT versus SEQ-7 escape populations. Differences
at four residues reached high statistical significance (Fisher’s
two tailed t test with Bonferroni correction for the number of
residues queried) (Table 3). Three examples are particularly
striking. Substitutions at residue 169 were present in 12% of
WT variants but only 0.5% of SEQ-7 variants. Sixteen percent
of WT variants had alterations at residue 207, while the lone
mutant detected among nearly 200 SEQ-7 variants analyzed
had a compensatory D225Y substitution, a residue known to
modulate HA receptor avidity/specificity (Stevens et al.,
2006). Conversely, none of the 200 hundred-plus WT mutants
had substitutions at residue 225, which was represented in
18% of SEQ-7 mutants.
We extended these findings by using H17-L10 to select
a repertoire of SEQ-8 escape mutants. Of escape variants,
12.5% and 10% exhibited alterations in residues 210 and 222,
while mutation in these residues comprises less than 1% of
WT and SEQ-7 escape repertoires. Seven other unique variants
are present at low frequency in the SEQ-8 escape repertoire.
repertoire is greatly affected by the exact HA sequence in the
globular domain. Substitution of even a single residue (E119K,
in the Cb epitope) between SEQ-7 and SEQ-8 has a major effect
on the repertoire of mutants that escape a mAb specific for
a different antigenic site (Ca).
Ab Escape by IAV HA Is Context Dependent
To better understand how HA sequence influences the escape
repertoire, we introduced D225Y into WT HA and S207P into
the SEQ-7 HA using the PR8 pDZ reverse-genetics plasmid
system (Quinlivan et al., 2005). The four viruses achieved
similar titers in MDCK cells (Figures 4A–4C). Each of the
mutants demonstrated reduced titers in eggs, however, with
SEQ-7-S207P demonstrating a 6-log10reduction in titer that
accounts for its absence from the H17-L2/H17-L10 escape
repertoire in egg grown virus stocks. The difference in growth
in egg versus MDCK cells is likely due to alterations in HA
By contrast, the effect of D225Y on PR8 growth in eggs,
though statistically significant, was relatively minor, and its
subdetectable frequency in virus stocks is due to another factor.
Table 2. mAbs Reactivity Parallels Functional pAb Recognition
Virus Number mAb +
Guinea PigRabbit Chicken
PR8182 = 100% 1,7501,7502,5601,280 3203201601,280
SEQ-3 ND*7007001280 ND80160 4080
SEQ-6ND*200 200320ND 4080 <1040
SEQ-10 7%110(6%) 110(6%)ND ND40 20<10 40
ND*, not done. WT or SEQ- mutants were tested for their binding to mAbs by indirect RIA and were scored for >10-fold decrease in affinity. By SEQ-12
all but four mAbs showed large affinity losses—the remaining four mAbs have low HI/VN activities. Viruses were also tested in standard HI assays
against pooled Balb/c mouse primary or secondary sera (following i.p. PR8 infection), ferret sera from three animals (post-i.n. infection), pooled guinea
pig sera (following i.n. infection), rabbit hyperimmune serum (following s.c. priming and boosting twice), and chicken serum. ND, not determined. Also
see Table S1.
Cell Host & Microbe
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As seen in Figures 4D and 4E, this substitution decreases the Ab
binding affinity of H17-L10 or H17-L2 for PR8 by only ?2-fold.
Although these mAbs bind equally or even more avidly to
SEQ-7, D225Y reduces mAb binding at least 1,000-fold, typical
for escape mutants (Frankel and Gerhard, 1979).
Based on these findings, we conclude that two important
factors sculpting HA mutation space, HA functionality and anti-
genicity, are exquisitely influenced by the existing sequence.
S207P illustrates an example of where an amino substitution
compromises the function of HA in the context of WT PR8 (but
not SEQ-7) in a host cell-dependent manner (growth in eggs
being severely compromised), while D225Y induces a major
antigenic alteration in the Ca antigenic site in the context of
SEQ-7, but not WT PR8.
One of the most puzzling features of HA antigenic drift is the
‘‘linear’’ nature of HA evolution (Ito et al., 2011; Pybus and
Rambaut, 2009). At any one time, isolates populating numerous
evolutionary branches cocirculate in humans. Over a relatively
short interval, selected isolates predominate to form the major
evolutionary tree. This is consistent with the idea that relatively
subtle mutations in HA confer large selective advantages.
Mathematical analysis and modeling provides enormous
insight into HA evolution, but relies on a limited appreciation
of the selective forces for amino acid substitutions as it focuses
on antigenicity and receptor binding based on the location of
the residue in defined antigenic or receptor binding sites
(Ferguson et al., 2003; Ito et al., 2011; Koelle et al., 2006;
Rambaut et al., 2008). A more complete understanding of HA
evolution will entail considering the influence of amino acid
substitutions acting at a distance on HA antigenicity and
receptor binding, as well as the effects of mutations on other
aspects of HA function, including membrane fusion activity
and interaction with NA (Hensley et al., 2011; Kryazhimskiy
et al., 2011) and other viral and host gene products (Tate
et al., 2011).
Here, to better understand HA evolution, we sequentially
selected virus with a series of mAbs until HA demonstrated a
physiologically significant decrease in binding affinities with
members of a very large panel of mAbs that neutralize the
parent virus. We find that 12 mutations are required to
‘‘completely’’ abrogate antigenicity by these criteria, though it
Seq 1 Seq 2Seq 3
Seq 7Seq 8
(fold change compared to PR8)
Seq 1Seq 2Seq 3Seq 4
Seq 5Seq 6Seq 7Seq 8
Seq 10Seq 11Seq 12
NA/HA by western
Figure 2. Sequential Variants Acquire Epistatic Changes in NA to Optimize Receptor Avidity
(A) NA activities of HAU-normalized viruses ± detergent to dissociate viral glycoproteins were determined using a small fluorogenic substrate.
(B) NA activities of PR8, SEQ-4, SEQ-8, and SEQ-12 normalized to the amount of NA protein in virions as determined by western blot.
(C) Ratios of NA/HA protein content in virions determined by immunoblotting.
(D) Receptor binding avidities of SEQ- variants were measured, determining the amount of RDE required to abrogate agglutination of turkey RBCs. Data are
represented as mean ± SEM.
Cell Host & Microbe
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318 Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc.
is important to recognize the escape is a continuous and not
quantal process; escape mutants will no longer escape given
sufficiently high concentrations of neutralizing Abs. Definitive
evidence that SEQ-12 effectively escapes immunity comes
from in vivo competition experiments, where in a single passage
in immune mice SEQ-12 outcompetes WT virus despite starting
as a minor population.
Notably, we encountered no obvious bottlenecks during the
sequential selection process, as mutants were obtained at
each step at typical frequencies. It is clear from the rate of IAV
evolution in nature and the very existence of 17 distinct subtypes
that HA is capable of accommodating a large variety of amino
acid substitutions and maintain not only function but also
a remarkably similar structure. This in itself is not unusual; nearly
all virus families demonstrate high sequence diversity yet main-
tain structure/function conservation in their attachment proteins,
when considering all the serotypes that have evolved. The
essential feature of antigenic plasticity is not the degree of diver-
sity but how rapidly diversity can be achieved. As proteins
mutate, they explore new fitness landscapes, and over an
extended period, high diversity may be achieved as epistatic
mutations restore function.
A critical question to be addressed in future studies is whether
other viral glycoproteins exhibit similar plasticity to HA. It will be
particularly interesting to compare HA to the functionally similar
paramyxovirus HN which demonstrates a similar frequency of
single escape variants (van Wyke Coelingh et al., 1987; Yewdell
and Gerhard, 1982) yet evolves much more slowly than HA in
Although variants were easily obtained at each selection step,
sequencing revealed a remarkable epistatic change that
occurred at the eighth selection step, where E119K was accom-
Figure 3. Locating H17-L2 and H17-L10
Three-dimensional model of HA rendered by
PyMOL software as a solid surface looking at top
and side of the trimeric molecule (using PR8 HA
crystal structure 1RVX). Amino acid substitutions
in sequential variants are labeled in blue. Escape
mutants of H17-L10 and H17-L2 that are present
in both PR8 and SEQ-7 repertoire are labeled
in red. Green shows substitutions exclusively in
PR8 escape repertoire, magenta shows substi-
tutions exclusively in SEQ-7 repertoire, and
yellow shows substitutions exclusively in SEQ-8
repertoire. See also Table 3 and Table S3.
panied by an amino acid substitution in
NA. The occurrence and fixation of the
NA mutation correlated with increased
avidity of HA for cellular receptors as
measured by binding to progressively
reverse mirror HA activity, as we have
previously noted (Das et al., 2011; Hens-
ley et al., 2011), but in this case the
opposite occurred: the NA mutation
reduced the amount of NA incorporated
per virion, while HA demonstrated increased capacity to bind
progressively desialylated RBCs. This emphasizes that the
assays we use to measure HA and NA function are imprecise
proxies for their natural functions, and caution against oversim-
plifying our concepts of HA and NA cooperativity, which ulti-
mately may include yet-undefined functions of each of these
Presumably, epistasis in NA occurs often during HA evolution,
and accounts for a substantial fraction of NA variation. NA-HA
epistasis suggests another possible reason for rapid antigenic
drift in IAV compared to other RNA viruses: IAV has evolved to
cushion alterations in HA function by compensatory changes in
NA. This process might be further facilitated by the segmented
nature of the IAV genome, which allows extremely rapid reas-
sortment and rescue by gene segments under conditions of
multiple infections (Nelson et al., 2008). Although the SEQ vari-
ants exhibited no coding changes in other genes, it is possible
that HA also interacts with other viral gene products, e.g. M1
or M2, that exert epistatic effects.
We show that HA gradually changes its antigenicity with each
selection step, indicating that the effects of amino acid substi-
tutions on antigenicity are highly local. This is expected from
the high structural conservation between WT and escape
mutant HAs (Knossow et al., 1984), and particularly between
widely divergent HA subtypes (Gamblin and Skehel, 2010). It
is important to note that it is not possible to categorically deter-
mine the minimal alterations in HA required for escape from
neutralizing Abs, since this is dependent on the exact compo-
sition and amounts of Abs present at the point of neutralization
in vivo. This will vary enormously based on genetic differences
between individuals, and particularly on their prior exposure to
various HAs present in vaccines and natural infections. Thus,
Cell Host & Microbe
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Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc. 319
given an immunodominant response to a single antigenic site
and a substitution that reduces the affinity of large majority of
Abs specific for the site, even a single substitution can poten-
tially provide some measure of escape (Jin et al., 2005; Natali
et al., 1984; Smith et al., 2004), though the Ab response would
likely have to be relatively weak (Cleveland et al., 1997; Cobey
and Pascual, 2011). We also note that it is likely that with the
proper mixture of mAbs specific for a single site, single amino
acid substitutions could be identified that have maximal effects
on site antigenicity, reducing the total number of mutations
required to reach the antigenic distance from WT HA achieved
It is important to appreciate the highly conditional nature of
antigenicity. We show that D225Y substitution in the context of
WT HA has a 2-fold effect on the avidity of two mAbs, while di-
minishing avidity more than 1,000-fold in the context of SEQ-7.
Extending these findings to epidemic viruses, E190D or Q197R
substitutions selected for H17-L10 neutralization resistance in
WT PR8 do not substantially reduce H17-L10 binding in the
context of A/Bellamy/42 (which possesses E190D) or A/BH/
1935 (which possesses A197R) (http://www.ncbi.nlm.nih.gov/
genomes/FLU/flubiology.html) (Gerhard et al., 1981).
This means that great care must be taken in extrapolating the
effects of amino acid substitutions on antigenicity; clearly
context can dictate the outcome. Indeed, the importance of
context is the critical finding of our study: the precise sequence
of HA influences the effects of amino acid substitutions on HA
antigenicity (a single amino acid change between SEQ-7 and
SEQ-8 changes the repertoire of escape mutants) and also
dictates the mutation space for further substitutions, as shown
bythe poor growthof the S207P in thecontext SEQ-7 replicating
in MDCK cells.
Given the large number of highly variable residues in HA that
can potentially influence the mutation fitness landscape, this
casts doubts on the possibility of confidently predicting HA
Table 3. Distinct WT versus SEQ-7 H17-L10 or H17-L2 Escape
H17-L10 H17-L2 H17-10 H17-L2H17-L10
Large panels of escape mutants were selected using PR8, SEQ-7, or
SEQ-8 in presence of saturating amounts of H17-L10 or H17-L2 Abs.
After sequencing, mutants were grouped based on the substitution in
HA.glyGenerates an additional glycosylation site (N-x-S/T, where x is
any amino acid except P). P value from Fisher’s two tailed t-test. With
a Bonferroni correction of 23 residues at which substitutions occur,
true significance at p < 0.05 must be corrected by dividing by 23,
which = 0.002. For detail of the substitutions in each escape mutant,
also see Figure 3 and Table S3.
aOnly present as a double mutant, likely to represent an epistatic substi-
tution to improve fitness of mAb escape.
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320 Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc.
evolution in nature, a problem exacerbated by context-specific
effects on antigenicity. All the more reason to focus efforts on
developing vaccines targeted to conserved antigenic sites on
HA (Han and Marasco, 2011; Kaur et al., 2011; Yewdell, 2011).
PR8 (originally obtained from Mount Sinai School of Medicine, New York, NY)
and mutants were grown in allantoic cavities of embryonated hen eggs and
were purified by differential centrifugation.
Hybridoma anti-HA Abs were produced and characterized as previously
described (Caton et al., 1982; Gerhard et al., 1981; Staudt and Gerhard,
1983; Yewdell, 1981).
Variants were selected by using the allantois-on-shell (AOS) culture system or
MDCK culture systems. Variant selection in AOS culture was done as
described before (Gerhard et al., 1981). MDCK cells were seeded in 96-well
plates (50,000 cells per well) and cultured for 16–24 hrs. Virus was serially
of an overneutralizing concentration of mAb was added to each well, and the
mixture was incubated at room temperature for 1 hr before adding to MDCK
cells. After 2 hr at 37?C, cells were washed with PBS and replenished with
200 ml of media supplemented with 1 mg/ml TPCK trypsin and the selecting
mAb. Cytopathic effect (CPE) wasmonitored 72–96 hr postinfection. Superna-
tants from CPE-positive wells (endpoint titer) were isolated for further amplifi-
cation and characterization. Variants frequencies were determined as
described elsewhere (Yewdell et al., 1986).
PR8 1.4 nM
PR8-D225Y 2.6 nM
SEQ7 0.8 nM
PR8 5.5 nM
PR8-D225Y 11.9 nM
SEQ7 4.3 nM
Figure 4. Antigenic Evolution Is Context
(A) TCID50titers of WT and mutant viruses rescued
(P0 passage) after eight-plasmid transfection in
(B) TCID50titers of WT and mutant viruses rescued
(P1-M passage) after amplifying in MDCK cells.
(C) TCID50titers of WT and mutant viruses rescued
(P1-E passage) after amplifying in MDCK cells. All
the experiments were performed in quadruplet,
and the p values were calculated using Prism
software (Student’s t test).
(D and E) Binding affinities of H17-L10 and H17-L2
to PR8, PR8-D225Y, SEQ-7, and SEQ-7-D225Y
were calculated by ELISA using detergent-dis-
rupted viruses. All the experiments were done in
quadruplicate, and dissociation constants were
calculated using Prism software. All avidities re-
ported demonstrated excellent fit for one site
binding with Hill slope curve fitting (R2values >
0.98). Data are represented as mean ± SEM.
See also Table S2.
RIAs were performed as previously described
(Gerhard et al., 1981) using detergent-disrupted
virus adsorbed to 96-well polyvinyl microtiter
plates and125I-labeled rabbit anti-mouse F(ab)02
to detect bound Ab.
night at 4?C with saturating amounts of virus in
allantoic fluid, then washed three times with PBS
containing 0.05% v/v Tween-20, and blocked with PBS with 7.5% FBS for
1 hr. Abs present in culture fluid or ascites fluids over a complete range of dilu-
tions from nondetectable to saturating binding were added for 2 hr at room
product was determined by ELISA plate reader.Ab concentrations were deter-
mined by competition ELISA using purified mAbs as standards. Ab avidities
excellent fit for one site binding with Hill slope curve fitting (R2values > 0.98).
HI Assay, Virus Neutralization Assays
HI assays were performed in round-bottom 96-well polystyrene microtiter
plates as previously described (Hensley et al., 2009). Virus neutralization
assays were performed as described (Hensley et al., 2009). Each assay was
repeated at least three times with three to five replicates per assay.
NA activity was determined as previously described (Hensley et al., 2011).
Purified virus doses were adjusted for NA levels as determined by ELISA (for
each virus, this corresponded to ?50–100 HAUs of virus/sample). Viruses
were diluted in assay dilution buffer (33 mM MES [pH 6.5], 4 mM CaCl2) with
200uMof 29-4-methylumbelliferyl-alpha-D-N-acetyneuraminic acid. Samples
were placed in black flat-bottom plates at 37?C, and OD (Ex = 365 nm; Em =
450 nm) readings were recorded every minute for 30 min. Data are expressed
were preincubated with 1% Triton-X to disrupt viral membranes. For some
experiments, results were normalized based on relative NA protein concentra-
tion determined by quantitative western blot.
Virion Protein Content Analysis
electrophoresis, and transferred to PVDF membranes. Blots were stained with
Cell Host & Microbe
Defining Antigenic Drift
Cell Host & Microbe 13, 314–323, March 13, 2013 ª2013 Elsevier Inc. 321
mouse anti-HA2 mAb RA5-22 and rabbit anti-NA C terminus pAb, both of
were then probed with IRDye 680 nm and 800 nm conjugated secondary Abs
(Li-Cor) and simultaneously visualized and quantitated on an Odyssey infrared
scanner using Image Studio v2.0 software (Li-Cor).
Viral RNA was isolated from MDCK supernatant (endpoint titrated viruses and
stocks) or allantoic fluid using QiAmp viral RNA mini kit (Qiagen) by using the
manufacturer’s protocol. cDNA was synthesized using a one-step reverse
transcriptase kit (Origene Technologies). Gene-specific primers (PR8 HA-F,
50-ATGAAGGCAAACCTACTGGTCCTG-30; PR8 HA1-R, 50-CTGCATAGCCT
GATCCCTGTT-30) amplified HA1, the PCR products were purified with
a QIAQuick PCR purification kit (Qiagen), and sequencing was performed
through a third-party sequencing service (MacrogenUSA) using dye-termi-
nator cycle sequencing system with an ABI sequencer (Perkin-Elmer).
Generation of Recombinant Viruses
The recombinant WT and mutant viruses were generated by cotransfection
of eight reverse-genetics plasmids containing the double-stranded DNA
toxin-free ultrapure plasmid for each gene segment (total 16 mg) was mixed
and transfected using calcium phosphate method to achieve ?90%–95%
transfection efficiency. The supernatants were collected 48 hr posttransfec-
tion and treated with 10 mg/ml tosylsulfonyl phenylalanyl chloromethyl ketone
(TPCK)-trypsin (Worthington, Lakewood, NJ) for 1 hr at 37?C. TPCK-trypsin-
treated viruses were either infected to overnight grown MDCK cells (P1-M
passage) or 10-day-old embryonated eggs (P1-E passage). Virus from 293T
cells, MDCK cells, and eggs was titered by TCID50in MDCK cells.
In Vivo Challenge Experiment
For vaccine stocks, allantoic fluid was incubated with 0.05% paraformalde-
hyde for 2 days at 4?C. Mice were injected intraperitoneally with 2,000 HAU
of inactivated influenza A viruses. Three weeks after vaccination, naive and
immunized mice were tail vein bled, and sera was RDE treated overnight at
37?C, inactivated by incubating at 56?C for 30 min, and tested for anti-IAV
Abs by HAI using turkey erythrocytes. Naive and vaccinated mice (five per
group) were anesthetized with isoflurane and infected intranasally with 104.8
TCID50units each of a mixture of PR8 and SEQ-12 viruses diluted in 50 ul
BSS + 0.1% BSA. Lungs isolated 2 days after infection were homogenized
and viral titers determined by endpoint dilution in MDCK cells. The relative
abundance of PR8 and SEQ-12 variants was determined by sequencing
a portion of the HA gene of plaque-cloned viruses from each mouse lung. All
mouse experiments were conducted in accordance with the guidelines of
the NIAID Institutional Animal Care and Use Committee.
The GenBank accession numbers for the WT and mutant influenza A virus (A/
PR/8/1934 H1N1) genome sequences (full length and partial) reported in this
paper are CY084006, CY084007, CY084008, CY084009, CY0840010,
CY105937, CY105938, CY105939, CY105940, CY105941, and CY105942.
Supplemental Information includes one figure and three tables and can be
found with this article at http://dx.doi.org/10.1016/j.chom.2013.02.008.
We thank Glennys Reynoso for providing outstanding technical assistance.
We thank Dr. Walter Gerhard for his generous gift of the large panel of mAbs
and viruses. We thank Dr. Adolfo Garcia-Sastre and Dr. David Wentworth for
kindly providing eight-plasmid reverse-genetics plasmid to rescue WT and
mutant viruses. We thank Dr. Zhiping Ye for providing ferret anti-sera to PR8
virus. The sequencing of the whole genome of the SEQ- variants was done
at JCVI, which is one of the Genomic Sequencing Center of Infectious
Diseases (GSCID) and has been funded with federal funds from the NIAID,
NIH, and Department of Health and Human Services under contract number
HHSN272200900007C. S.R.D. is supported by JCVI internal start-up fund.
J.W.Y. and J.R.B. are generously supported by the Division of Intramural
Research, NIAID. S.R.D., S.E.H., W.L.I., G.R., J.R.B., and J.W.Y. conceived
of and designed experiments. S.R.D., S.E.H., W.L.I., C.B.B., A.S., M.G.D.,
G.R., and J.S.G. performed experiments. S.R.D., S.E.H., W.L.I., C.B.B., A.S.,
M.G.D., G.R., J.R.B., and J.W.Y. analyzed data. S.R.D. and J.W.Y. wrote the
Received: May 4, 2012
Revised: December 4, 2012
Accepted: February 20, 2013
Published: March 13, 2013
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