Fitness costs limit influenza A virus hemagglutinin
glycosylation as an immune evasion strategy
Suman R. Dasa,b,1, Scott E. Hensleya,2, Alexandre Davida, Loren Schmidta, James S. Gibbsa, Pere Puigbòc, William L. Incea,
Jack R. Benninka, and Jonathan W. Yewdella,3
aLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;bEmory
Vaccine Center, Emory University, Atlanta, GA 30322; andcNational Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, MD 20894
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved October 21, 2011 (received for review June 16, 2011)
Here, we address the question of why the influenza A virus
hemagglutinin (HA) does not escape immunity by hyperglycosyla-
tion. Uniquely among dozens of monoclonal antibodies specific for
A/Puerto Rico/8/34, escape from H28-A2 neutralization requires
substitutions introducing N-linked glycosylation at residue 131 or
144 in the globular domain. This escape decreases viral binding to
cellular receptors, which must be compensated for by additional
substitutions in HA or neuraminidase that enable viral replication.
Sequence analysis of circulating H1 influenza viruses confirms the in
vivo relevance of our findings: natural occurrence of glycosylation at
residue 131 is always accompanied by a compensatory mutation
known to increase HA receptor avidity. In vaccinated mice chal-
lenged with WT vs. H28-A2 escape mutants, the selective advantage
conferred by glycan-mediated global reduction in antigenicity is
trumped by the costs of diminished receptor avidity. These findings
show that, although N-linked glycosylation can broadly diminish HA
antigenicity, fitness costs restrict its deployment in immune evasion.
antigenic drift|viral evolution
response. Immune evasion is primarily based on the protean
capacity of the HA glycoprotein to accommodate amino acid
substitutions that modify antigenicity and modulate receptor
avidity. HA is a homotrimer that attaches virus to terminal sialic
acid residues on target cells to initiate the infectious cycle (1).
Antibodies (Abs) that interact with the globular HA domain
sterically block attachment and neutralize viral infectivity (2).
Sequencing RNA of escape mutants resistant to neutralization
by individual monoclonal Abs (mAbs) revealed that mutants
typically possess a single nucleotide change generating an amino
acid substitution in the globular domain (3). Localization of the
amino acid alterations in the crystal structure of the HA of A/
Puerto Rico/8/34 (PR8) clearly shows the presence of five dis-
tinct antigenic sites: Sa and Sb are located at the tip of the
globular domain, and Ca1, Ca2, and Cb are located more toward
the stem of H1 HA (3). This method of epitope mapping, al-
though indirect, is much more rapid and robust than other
methods, and it is still capable of providing a reasonable physical
definition of the relevant epitope (4).
The frequency of mutants that escape individual neutralizing
anti-HA mAbs is typically in the range of 10−4–10−6(5). Variants
capable of escaping selection mAbs specific for nonoverlapping
epitopes occur at a frequency of ∼10−10, consistent with two
independent point mutations (6). The low frequency of mutants
with multiple amino acid substitutions raises the question of how
IAVs evolve antigenically in man. Do individuals make biased
antibody responses enabling selection of single point mutants, or
do rare, multiply substituted mutants arise based on the very
large aggregate virus populations among infected humans.?
Acquisition of N-linked glycosylation sites near antigenic sites
represents another potential mechanism for IAV to escape an-
tibody neutralization, because the large size of oligosaccharides
nfluenza A virus (IAV) remains an important human patho-
gen, largely because of its ability to evade the humoral immune
can sterically prevent Ab access to its epitope. HIV gp160 pro-
vides a clear example of hyperglycosylation as an effective im-
mune escape mechanism (7). Interestingly, although the H3 HA
has gradually gained glycosylation sites in the globular region,
H1 HA circulating for a similar period in a similarly large pop-
ulation has acquired far fewer sites (8). Moreover, the H2 HA
during its 10 y of evolution in humans maintained a lone glyco-
sylation site in the globular domain.
The limited addition of glycosylation sites suggests a high se-
lection cost. Here, we provide compelling evidence for this
conclusion by studying the ability of PR8 to escape neutralization
of the H28-A2 mAb [designated Cx8 in the work by Gerhard
et al. (9)]. This IgM mAb, generated from a BALB/c mouse
immunized with infectious influenza, shows a number of unique
properties, including the ability to select viruses that only escape
neutralization through acquisition of an N-linked glycosylation
site in the globular domain.
H28-A2: A Unique mAb. H28-A2, one of hundreds of PR8 HA-
specific mAbs generated by the Gerhard laboratory, has a num-
ber of unique properties. First, unlike dozens of other anti-PR8
mAbs tested, the H28-A2 mAb selects escape mutants at a fre-
quency expected (5) for a double simultaneous mutation (<10−9.12)
(Table 1). Second, consistent with this property, H28-A2 alone
among hundreds of neutralizing mAbs shows little change in
binding affinity to a panel of more than 40 escape mutants with
amino acid substitutions distributed among the five antigenic
sites (3). Third, when one of the H28-A2 (termed Ab O in the
Gerhard laboratory’s escape variant nomenclature, where mutants
were named after their selecting Ab) escape mutants selected in
the allantois on shell system (OV1) was tested for binding to
a 220-member panel of HA-specific mAbs, it paradoxically
showed reduced binding to a remarkably large fraction of mAbs
(71%), including many mAbs that map to each of the four anti-
genic sites in the globular domain (Table 2).
The unique properties of H28-A2 are not related to unusual
gene use (10) or unusually high affinity. Affinity measurement by
ELISA performed with intact virus revealed a typical affinity
observed for HA-specific mAbs, with a KAof 1.1 × 109(Table 3).
Author contributions: S.R.D., J.R.B., and J.W.Y. designed research; S.R.D., S.E.H., A.D., L.S.,
J.S.G., P.P., W.L.I., and J.W.Y. performed research; S.R.D. contributed new reagents/
analytic tools; S.R.D., S.E.H., A.D., L.S., J.S.G., P.P., W.L.I., J.R.B., and J.W.Y. analyzed data;
and S.R.D., J.R.B., and J.W.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1Present address: Infectious Diseases Group, J. Craig Venter Institute, Rockville, MD 20852.
2Present address: Immunology Program, The Wistar Institute, Philadelphia, PA 19130.
3To whom correspondence should be addressed. E-mail: email@example.com.
See Author Summary on page 20289.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 20, 2011
| vol. 108
| no. 51
Escape mutants showed a decrease of affinity of >10,000-fold to
H28-A2 (Table 3).
H28-A2–Resistant Mutants Exhibit a Unique Escape Strategy. RNA
sequencing of two H28-A2 escape mutants (OV1 and OV2)
immediately explained their low frequency and high degree of
escape. Each mutant possessed a common nucleotide mutation
encoding a predicted substitution of K to N at position 144 (H3
numbering system based on PR8 HA crystal structure 1RVX)
and generating a potential N-linked glycosylation site at position
144, because position 146 encodes S. Importantly, each variant
had an additional mutation encoding an amino acid substitution:
D225G for OV1 and N193K for OV2 (Fig. 1).
We extended these findings to the Madin Darby canine kidney
(MDCK) system of escape mutant selection. Selection with
H28-A2 generated an escape mutant (OV3) at an extremely low
frequency (10−10.66). RNA sequencing once again revealed two
nonsynonymous mutations encoding the common K144N sub-
stitution accompanied by a P186S substitution.
Like virtually all HAs, the PR8 HA possesses glycosylation
sites at or near stem residues 15, 26, 289, 483, and 542 (8). Al-
though most contemporary H1 HAs possess glycosylation sites at
91, 129, or 162, PR8 lacks glycosylation sites in the globular
domain. Locating residue 144 in the H1 HA structure reveals
that it is present on the solvent-exposed surface, where it could
easily accommodate the addition of an oligosaccharide (Fig. 1).
To validate glycosylation at the introduced site, we purified
WT PR8, OV1, OV2, and OV3 and single escape mutants with
identical alterations to the second site mutations observed in the
OV variants (D225G, N193K, and P186S) (Fig. 2A). A mobility
shift in the HA1 chain consistent with additional glycosylation at
N144 was observed in SYPRO Ruby-stained reducing gels and
through immunoblotting with the anti-HA1 mAb CM-1.
After removal of oligosaccharides with PNGase F, all HA1
chains migrated with the same mobility, clearly showing that the
oligosaccharide attachment site generated by the K144N sub-
stitution is used (Fig. 2B).
Glycosylation at Position 144 Requires Compensatory Substitutions.
The bulk of an N-linked oligosaccharide, in conjunction with the
central location of the 144 residue amid the antigenic sites in the
globular domain (Fig. 1), provides an obvious steric explanation
for the dramatically diminished antigenicity of H28-A2 escape
mutants. It seemed likely, therefore, that the second mutations
present in the escape mutants are necessary to compensate for
functional deficits caused by glycosylation at position 144.
We confirmed this hypothesis by generating K144N recombi-
nant virus by the eight-plasmid reverse genetic system. Imme-
diate sequencing of virus from 293T culture supernatants post-
transfection showed only the intended K144N substitution.
However, as soon as we amplified this virus in eggs or MDCK
cells, compensatory mutations appeared. Sequencing cloned egg
virus revealed D225G or I194L substitutions. MDCK-propa-
gated viruses revealed three types of substitutions: (i) alterations
of N144 to Y or S, removing the glycosylation site, (ii) com-
pensatory changes in HA, P186S, or G156E, and most remark-
ably, (iii) single or double substitutions in NA (L22P, G27R,
P139S, P231L, and V273M with or without N434T; PR8 NA
numbering). The latter result confirmed our recent finding that
antigenic escape mutants in HA that limit fitness can be rescued
by NA amino acid substitutions (11).
We confirmed the compensatory nature of second site sub-
stitutions (D225G, N193K, and P186S) by using H28-A2 in the
MDCK cell system to select escape mutants from PR8 mutants
with substitutions in the defined compensatory sites. Now, vari-
ant frequencies occurred within the range typical of single point
mutants: 10−5.56for D225G and 10−4.21for P186S. H28-A2 es-
cape mutants from N193K mutants occurred at a lower fre-
Table 1. Frequencies of H28-A2 escape mutants
Virus + antibody Variant frequencyMutations in HA
WT + 1 mAb
WT + mix of 2 mAb
WT + H28-A2 (egg)
WT + H28-A2 (MDCK)
D225G + H28-A2 (MDCK)
N193K + H28-A2 (MDCK)
P186S + H28-A2 (MDCK)
K144N + D225G (OV1) K144N N193K (OV2)
K144N + P186S (OV3)
K144N or N133T
The frequency of escape mutants was determined as described (5).
Table 2.Antigenic mapping of OV1
Sa (23 mAbs)
Sb (90 mAbs)
Ca (1 + 2; 43 mAbs)
Cb (64 mAbs)
Total (220 mAbs)
A large panel (220 mAbs) of mAbs with defined epitopes as indicated was
used to determine the antigenicity of OV1 through radioimmunoassay as
described (9). The percent of mAbs specific for each site showing major
(more than fourfold) or moderate (two- to fourfold) decreases in avidity is
given by top and bottom (in parentheses) values, respectively.
Table 3.Interaction of mAbs with mutants
KA(cell staining)KA(ELISA)HI (Turkey RBCs)
1.9 × 109
<8.3 × 106
<8.3 × 106
<8.3 × 106
1.4 × 109
4.3 × 108
2.4 × 108
<8.3 × 106
<8.3 × 106
6.3 × 108
5.1 × 107
<8.3 × 106
<8.3 × 106
<8.3 × 106
<8.3 × 106
1.1 × 109
<8.3 × 106
<8.3 × 106
<8.3 × 106
1 × 109
2.7 × 108
2.3 × 108
<8.3 × 106
<8.3 × 106
5.5 × 108
1.9 × 108
<8.3 × 106
<8.3 × 106
<8.3 × 106
<8.3 × 106
Avidity values were determined either by staining 5 h postinfected cells or
ELISA using intact virus coated on plates.
| www.pnas.org/cgi/doi/10.1073/pnas.1108754108 Das et al.
quency (10−7.2), likely attributable to poor adaptation of these
double mutants to MDCK cells because of fitness differences
between the various compensatory substitutions.
Sequencing the sequentially selected viruses revealed the ex-
pected but also a surprise. Although all of the mutants obtained
from N193K or P186S stocks possessed the expected K144N
substitution, only 50% of D225G mutants possessed this sub-
stitution. Remarkably, the other 50% possessed an N133T sub-
stitution, now creating a glycosylation site at N131, located just
north of position 144 and very close to the receptor binding site
on the HA solvent-accessible surface (Fig. 1). Biochemical
analysis of a selected D225G N133T double mutant confirmed
glycosylation at position 131 (Fig. 2).
Together, these findings clearly show that escape from H28-
A2 neutralization requires two events: introduction of a glyco-
sylation site at residue 144 or 131 and substitution at residue 186,
193, or 225 (or various NA residues) to compensate for negative
effects of glycosylation on viral fitness.
Effect of Escape Mutations on H28-A2 Affinity. All escape mutants
with introduced glycans at HA residue 144 or 131 exhibit at least
1,000-fold decreased avidity for binding to H28-A2 determined
either by ELISA with virus as immunoabsorbent or flow cytom-
etry of infected cells (Table 3), showing that glycosylation alone
in this region enables immune escape. Either N193K or D225G
alone diminishes H28-A2 avidity, and the combination of K144E
and D225G decreases affinity more, supporting the contribution
of both of these residues to binding. The importance of residue
144 is also shown by the moderate effect of K144Y and K144S
substitutions on H28-A2 avidity. HI titers paralleled the ELISA
data, showing the functional consequences of diminished binding
Together, these findings show that H28-A2 binding is only
completely blocked by glycosylation in the region near the re-
ceptor binding site (RBS) and that substitutions in residues
surrounding the RBS have modest but clear effects on binding,
and they are likely to define the epitope.
HA Glycosylation Decreases Receptor Binding. Residues 144 and 131
are in relatively close proximity to the RBS, and it is expected
based on numerous studies that glycosylation at these residues
will reduce HA receptor avidity (12–14). Indeed, we reported
that the compensatory amino acids observed in the OV variants
increase HA receptor avidity (15, 16).
To measure HA receptor avidity, we determined the capacity
of viruses to agglutinate human or turkey erythrocytes treated
with increasing amounts of soluble bacterial neuraminidase [re-
ceptor-destroying enzyme (RDE)] (Fig. 3). This finding con-
firmed the ability of D225G, N193K, and P186S, all located in
close proximity to the RBS (Fig. 1), to increase viral receptor
avidity. Importantly, for each of these substitutions, glycosylation
at residue 144 or 131 reduced the avidity to lower than WT
levels. Also, glycosylation alone at K144N (with compensatory
mutations in NA that do not affect the RDE assay) or N133S
(which also creates a predicted glycosylation site at residue 131)
reduces HA receptor avidity.
From these findings, we infer that glycosylation near the RBS
interferes with receptor binding to the extent that compensatory
mutations that increase avidity or modulate NA function are
required to restore viral fitness.
Evidence of HA Compensatory Mutations with Additional Glycosylation
in Circulating Viruses. To relate these findings to natural IAV
evolution in humans, we analyzed 1,640 full-length H1 HA
sequences from human viruses downloaded from the National
Center for Biotechnology Information (NCBI) influenza virus
resource. The NetNGlyc prediction of glycosylation sites (Asn-
Xaa-Ser/Thr, where Xaa is any amino acid except Pro) in the
side of the trimeric molecule (using PR8 HA crystal structure 1RVX). Amino acid substitutions in escape mutants are indicated by color and label. In Z1, which
zooms in on the receptor binding site (RBS), the two glycosylation sites created by mutations, located at residues 144 (direct introduction of N) and 131
(mutation at residue 133 (red) creates a site for existing N) are shown in pink. Glycosylation at these residues causes global changes in antigenicity and also
incurs fitness costs that must be compensated for by epistatic substitutions (at the yellow residues) for virus survival.
Locating amino acid substitutions in H28-A2 escape mutants. 3D model of HA rendered by PyMol software as a solid surface looking at the top and
Das et al.PNAS
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globular domain reveals the nonrandom distribution of probable
glycosylation sites at nine locations. None of the isolates possess
a glycosylation site at position 144, confirming the selection
costs of glycosylation at this position in natural H1 evolution
Ninety-eight isolates, however, are predicted to be glycosy-
lated at position 131 (Table S1). Yearwise distribution of gly-
cosylation at position 131 showed a nonrandom distribution,
showing that it was not immediately supplanted by other strains
(Fig. S1). All of the isolates with glycosylation sites at position
131 possess the P186S substitution (Table S1), which increases
receptor avidity and has minimal effects on HA antigenicity (15).
Taken together, these findings support the conclusion that
glycosylation at position 144 incurs enormous fitness costs that
have yet to be surmounted in H1 evolution, whereas glycosylation
at position 131 is disfavored but can exist in circulating strains
with compensatory mutations that restore HA receptor avidity.
HA Avidity Trumps Antigenicity in Escape from Polyclonal Abs. Given
the dramatic effect of position 144 glycosylation on HA antige-
nicity (Table 1), it would be expected that the H28-A2 escape
mutants would show robust escape from a polyclonal Ab. In fact,
when tested in hemagglutination inhibition (HI) assays using
pooled sera from outbred Swiss mice immunized with PR8,
glycosylation had either a minor positive effect on escape (D225G
vs. K144N, D225G or N133T, D225G) or remarkably, the op-
posite effect on escape (N193K vs. K144N, N193K; P186S vs.
K144N, P186S) (Fig. 4A).
We extended these findings to an in vivo infection model by
immunizing mice with PR8, challenging them with WT, P186S,
or K144N, P186S viruses, and measuring viral lung titers 2 d
postinfection. In naïve mice, all three viruses replicated to sim-
ilar titers (Fig. 4B). Vaccination completely prevented replica-
tion of WT virus but only reduced replication of P186S by 10-
fold, similar to what we previously reported (16). By contrast,
mice were completely protected against K144N, P186S infection,
despite the enormous antigenic escape associated with glycosyl-
ation at position 144.
These findings point to the important conclusion that receptor
avidity can be a more important factor than antigenicity in es-
caping from neutralizing antibodies, which also limits the use of
glycosylation as a means of HA immune escape because of its
attendant reduction in avidity.
The resistance of H28-A2 to immune escape by single amino
acid substitutions in a highly variable HA domain capable of
accepting numerous different substitutions is, to our knowledge,
unprecedented among anti-HA mAbs. H28-A2 derives from
a BALB/c mouse immunized i.p. with infectious PR8 and
boosted i.v. 1.5 y later with PR8 3 d before fusion. Three other
mAbs characterized in detail from the same fusion are specific
for the Sb site, which is likely immunodominant in mice (16, 17).
DNA sequencing of H28-A2 heavy and light chains revealed that
it is closely related to six other mAbs derived from four in-
dividual BALB/c mice, all of which share VH 7183 and Vk 24
family genes (10). H28-A2 and three of six mAbs share the un-
usual property of binding with high affinity to the Denver 1957
H1 isolate (18). Examination of the reactivity patterns of the
sites. Egg-grown viruses were purified and analyzed by SDS/PAGE. (A) SYPRO
Ruby staining or immunoblotting by anti-HA antibody (CM1) shows a shift in
HA mobility because of additional glycosylation, which was confirmed by
near identical mobilities following PNGase F deglycosylation (B).
Biochemical confirmation of N-linked glycosylation at introduced
enzymes (RDEs) before the addition of viruses indicated to determine the relative amounts of terminal sialic acid required for agglutination.
Glycosylation diminishes receptor binding avidity. (A) Human or (B) turkey RBCs were treated with graded concentrations of receptor-destroying
| www.pnas.org/cgi/doi/10.1073/pnas.1108754108 Das et al.
Denver-reactive VH7183/Vk24+ mAbs reveals large affinity
losses to OV1 and mutants with amino acid substitutions at 193
and 145. These residues along with 225, where a D to G sub-
stitution reduces H28-A2 avidity by fivefold (Table 3), define a
binding footprint consistent with the known interaction surface
of antibodies with HA (Fig. 1). This proposed location of the
H28-A2 epitope would explain the predicted steric effects of
glycosylation at position 144 or 131 on H28-A2 binding. In-
terestingly, H28-A2 sits squarely atop the sialic acid binding
site, which could be the key to its resistance to single amino
The central location of residue 144 on the HA also provides
a ready explanation for how glycosylation of this residue can
reduce the binding of mAbs specific for each of the antigenic
sites. This finding extends previous findings that glycosylation
modulates HA antigenicity (12, 13, 19) and immunogenicity (20).
The low frequency of H28-A2 escape mutants, in conjunction
with their special nature in generating a glycosylation site, con-
clusively indicates that HA is incapable of generating viable
mutants with single amino acid substitutions that enable escape
from H28-A2. Explanation for this remarkable property will
likely require crystallographic analysis of H28-A2 complexed
with HA, perhaps compared with the Denver-reactive VH7183/
Vk24+ Abs, which likely define a highly similar epitope but are
sensitive to individual side chain substitutions. Until then, we can
speculate that structural limitations in the epitope region com-
bine with unusual features in H28-A2 to limit escape. Un-
derstanding the features of antibodies that resist antigenic drift is
of obvious practical importance in devising vaccines for viruses
like IAV and HIV, where antigenic variation greatly impacts
vaccine effectiveness. It is important to note that there might be
significant differences between mouse and human anti-HA Ab
repertoires because of differences in germ-line Ab genes, so-
matic mutation tendencies, or frequent reexposure of humans to
evolving IAV strains.
Glycosylation at residue 144 is not observed among H1 iso-
lates, confirming its fitness costs in the real world evolution of
IAV in humans. By contrast, residue 131 is likely glycosylated in
a number of strains, including A/Melbourne/35, which shows
greatly reduced binding of H28-A2 (9). The glycosylation site in
Mel is created by insertion of a triplet between residues 132 and
133 in PR8, and it is accompanied by 15 other amino acid sub-
stitutions in the globular domain and 18 other HA substitutions.
This finding shows that, for some glycosylation sites that protect
antigenic regions, the H1 HA is fully capable of acquiring
compensatory mutations that mitigate the functional con-
sequences of glycosylation. However, in over 60 y of circulation
in human populations, the H1 HA maximally encodes only three
potential glycosylation sites in the globular domain (8). Over
a similar time frame, the H3 HA maximally encodes six sites,
indicative of the difficulty of the H1 HA to maintain function
with increasing glycosylation. It will be of interest to observe the
evolution in man of the swine origin IAV, which possesses but
a single glycosylation site in the globular domain. Notably, H2
viruses maintained a single glycosylation site in their 11-y cir-
culation from 1957 to 1968, pointing to a high cost of glycosyl-
ation, which was shown in the work by Tsuchiya et al. (13).
Glycosylation at position 144 reduced HA receptor affinity,
consistent with prior studies documenting a deleterious effect of
glycosylation on HA binding to sialic acid receptors (12–14). The
effect of position 144 glycosylation on receptor avidity allowed us
to test our recent findings regarding the importance of HA re-
ceptor avidity in antigenic drift (16). Despite the enormous effect
of glycosylation on overall antigenicity as measured by a signifi-
cant decrease in binding affinity of 71% of a large and diverse
mAb panel, the decrease in receptor affinity incurred by glyco-
sylation at position 144 offset the evolutionary advantage con-
ferred by antigenic escape.
This finding hammers home the point that receptor affinity
can play a dominant role in escape from neutralizing antibodies.
HA glycosylation is highly likely to interfere with receptor
binding in a manner that must be compensated for by additional
mutations, creating a fitness barrier to accumulating glycosylation
sites and providing a ready explanation for the paucity of oli-
gosaccharides on HA compared with other viral receptor pro-
teins (e.g., HIV gp160).
This discussion is relevant to the recent description of mAbs
that bind highly conserved epitopes in the stem region of HA,
which allows them to neutralize nearly all isolates within the H1
subtype and even between H1, H2, and H5 subtypes (21).
Binding to H3 HA, however, is likely blocked by the presence of
an N-linked glycan absent on H1/H2/H5 HAs (22, 23), raising
the possibility of glycan-based escape from immunization strat-
egies aimed at inducing stem-reactive mAbs. Our findings sug-
gest that the costs of HA glycosylation can be sufficient to delay,
if not preclude, its deployment as an immune escape strategy.
Virus, Antibodies, and Mice. WT and escape mutants of PR8 (H1N1; Mt. Sinai
strain) were propagated in 10-d embryonated chicken eggs (CBT Farms).
pAb escape (HI)
a glycosylation site at residue 144 diminishes HI titer. Each experiment was repeated three times with quadruple samples, and one representative experiment
is presented here. Similarly, in mouse infections (B), addition of a glycosylation site at residue 144 to the adsorptive mutant S186P abrogates escape from anti-
HA Abs induced by vaccination as shown by recovery of infectious virus from lungs. In each group, five mice were used per experiment, and representative
data are presented of three separate experiments.
Receptor binding modulates immune escape in vitro and in vivo. HI assays (A) using pooled mouse primary anti-PR8 antisera show that introduction of
Das et al.PNAS
| December 20, 2011
| vol. 108
| no. 51
Hybridoma anti-HA antibodies were produced and characterized as pre-
viously described (9).
Swiss mice were purchased from Charles River, and C57BL/6 and BALB/c
mice were purchased from Taconic. Mice were maintained under specific
pathogen-free conditions, and all procedures involving mice were approved
by the National Institute of Allergy and Infectious Disease animal care and
Avidity Measurements. Virus ELISA. Ninety-six–well plates (4HBX; Immunlon)
were coated overnight at 4 °C with saturating amounts of virus in allantoic
fluid, washed three times with PBS containing 0.05% vol/vol Tween-20, and
blocked with PBS with 7.5% FBS for 1 h. Antibodies present in culture fluid
or ascites fluids over a complete range of dilutions from nondetectable to
saturating binding were added for 2 h at room temperature. After washing,
100 μL TMB substrate (KPL) were added. The reaction was stopped by the
addition of 50 μL 0.1 N HCl, and the amount of product was determined by
an ELISA plate reader. Antibody concentrations were determined by com-
petition ELISA using purified mAbs as standards. Antibody avidities were
determined using Prism software, and all avidities reported showed excel-
lent fit for one-site binding with Hill slope curve fitting (R2values > 0.98).
Cell staining. MDCK cells were infected at a multiplicity of infection of five
with either WT or mutant viruses for 5 h in suspension. Cells were washed
with BSS-PBS supplemented with bovine serum albumin and incubated at
4 °C with mAbs over a range of dilutions from background to saturating
binding for 1 h, which was followed by FITC-labeled secondary Abs. Cells
were analyzed live at 4 °C by flow cytometry, and mean fluorescent intensities
for each concentration of mAbs were used to calculate avidities using Prism
HA, HI, and Virus Neutralization Assays. HA, HI, and virus neutralization assays
were performed as described (16). Each assay was repeated at least three
times with three to five replicates per each assay.
Variant selection. Variants frequencies were determined either as described (5)
or using a modified protocol with MDCK cells. MDCK cells were seeded in 96-
well plates (50,000 cells/well) and cultured for 16–24 h. Virus was serially
diluted 10-fold (10−1–10−8) at 50 μL/well in a parallel 96-well plate. An equal
volume of an overneutralizing concentration of mAb was added to each
well, and the mixture was incubated at room temperature for 1 h before
adding to MDCK cells. After 2 h at 37 °C, cells were washed with PBS and
replenished with 200 μL media supplemented with 1 μg/mL TPCK trypsin and
the selecting mAb. Cytopathic effect (CPE) was monitored 72–96 h post-
infection. Supernatants from CPE-positive wells (endpoint titer) were iso-
lated for additional amplification and characterization.
RNA sequencing. Viral RNA was isolated from MDCK supernatant (endpoint
titrated viruses and stocks) or allantoic fluid using QiAmp Viral RNA Mini Kit
(Qiagen) using the manufacturer’s protocol. cDNA was synthesized using a
one-step reverse transcriptase kit (Origene Technologies). Gene-specific
primers (PR8 HA-F, 5′-ATGAAGGCAAACCTACTGGTCCTG-3′; PR8 HA1-R, 5′-
CTGCATAGCCTGATCCCTGTT-3′) amplified HA1, and the PCR products were
purified with a QIAQuick PCR purification kit (Qiagen); sequencing was per-
formed through a third-party sequencing service (MacrogenUSA) using dye
terminator cycle sequencing system with an ABI sequencer (Perkin-Elmer).
In vivo challenge experiment. For vaccine stocks, allantoic fluid was treated with
paraformaldehyde (1:864 dilution) for 2 d at 4 °C. Mice were injected in-
traperitoneally with ∼100–500 hemagglutination units inactivated IVA; 10 d
after vaccination, mice were retroorbitally bled, and the erythrocytes were
removed using serum gel tubes (Sarstedt). Serum was inactivated by in-
cubating at 56 °C for 30 min and tested for anti-IAV antibodies by HI. Naïve
and vaccinated mice (five animals per group repeated three times) were
anesthetized with isoflurane and infected intranasally with 105tissue culture
50% infectious dose (TCID50) units of WT and mutant viruses diluted in 50 μL
PBS. Lungs isolated 2 d after infection were homogenized, and viral titers
were determined by endpoint dilution in MDCK cells.
Source of Sequences, Prediction of N-Glycosylation Sites, and Multiple Sequence
Alignment. A set of 1,640 full-length H1N1 HA sequences (human hosts from
virus resource at NCBI (http://www.ncbi.nlm.nih.gov/genomes/FLU) (8). The
NetNGlyc 1.0 web server (http://www.cbs.dtu.dk/services/NetNGlyc) was used
to predict N-glycosylation sites (Asn-Xaa-Ser/Thr, where Xaa is any amino acid
except Pro) of all HA sequences; a positive was scored when the jury returned
a + score. According to NetNGlcy, 76% of positive-scored sequons are mod-
ified by N-glycans, with a bias to Thr-containing sequons. All HA sequences of
H1N1 were aligned in a single common alignment using the program Muscle
with default parameters (8).
ACKNOWLEDGMENTS. We thank Glennys Reynoso for providing outstand-
ing technical assistance. This work was generously supported by the Division
of Intramural Research National Institute of Allergy and Infectious Diseases
(NIAID). S.R.D. is currently supported by National Institutes of Health/NIAID
Grant U19-AI057266 with American Recovery and Reinvestment Act Supple-
ment Funding Grant U19 AI057266-06S2.
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