JOURNAL OF VIROLOGY, Dec. 2007, p. 12816–12826
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 23
Type- and Subcomplex-Specific Neutralizing Antibodies against
Domain III of Dengue Virus Type 2 Envelope Protein
Recognize Adjacent Epitopes?
Soila Sukupolvi-Petty,1† S. Kyle Austin,2† Whitney E. Purtha,2Theodore Oliphant,3Grant E. Nybakken,2
Jacob J. Schlesinger,4John T. Roehrig,5Gregory D. Gromowski,6Alan D. Barrett,6
Daved H. Fremont,2and Michael S. Diamond1,2,3*
Departments of Medicine,1Pathology & Immunology,2and Molecular Microbiology,3Washington University School of Medicine,
St. Louis, Missouri; Department of Medicine, University of Rochester, Rochester, New York4; Centers for Disease Control and
Prevention, Fort Collins, Colorado5; and Center for Biodefense and Emerging Infectious Diseases, Sealy Center for
Vaccine Development, Institute for Human Infections and Immunity, and Department of Pathology,
University of Texas Medical Branch, Galveston, Texas6
Received 28 February 2007/Accepted 30 August 2007
Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral
envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the
lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in
animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for
individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of
DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs
that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with
neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered
at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly
neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized
DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305,
K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted
with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a
conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define
adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and
cross-reactive antibodies with different neutralizing potentials.
Dengue fever (DF), the most prevalent arthropod-borne
viral illness in humans, is caused by dengue virus (DENV). The
four serotypes of DENV are transmitted to humans primarily
by the mosquitoes Aedes aegypti and Aedes albopictus. DENV is
a member of the Flaviviridae family and is related to the viruses
that cause yellow fever and the Japanese, St. Louis, and West
Nile encephalitides (8). Infection by DENV causes a spectrum
of clinical disease, ranging from an acute, debilitating, self-
limited febrile illness (DF) to a life-threatening hemorrhagic
and capillary leak syndrome (dengue hemorrhagic fever/den-
gue shock syndrome). At present, no approved antiviral treat-
ment or vaccine is available, and therapy is supportive in na-
ture. DENV causes an estimated 25 to 100 million cases of DF
and 250,000 cases of dengue hemorrhagic fever per year world-
wide, with 2.5 billion people at risk for infection (27, 48).
DENV is an enveloped virus with a single-stranded, positive-
sense RNA genome (11). The 10.7-kilobase genome is trans-
lated as a single polyprotein, which is then cleaved into three
structural proteins (C, prM/M, and E) and seven nonstructural
(NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
NS5) by virus- and host-encoded proteases. The 500-Å DENV
mature virion has a well-organized outer protein shell, a 50-
Å-thick lipid membrane bilayer, and a less-defined inner nu-
cleocapsid core (37, 79). The icosahedral scaffold consists of
180 E and 180 M protein monomers arranged in a repeating
pattern that lacks the predicted T?3 quasisymmetry (37, 78).
The immature virion, which lacks cleavage of the prM protein,
has a rough surface with 60 spikes (79), whereas the mature
virion has a smooth surface. X-ray crystallographic analyses of
the soluble ectodomains of the E proteins from tick-borne
encephalitis virus and DENV demonstrated a dimeric assem-
bly, with each subunit containing three domains (46, 59, 60).
Domain III (DIII), which adopts an immunoglobulin-like fold,
is believed by some to contain a cell surface receptor recogni-
tion site (3, 60, 74, 77). Recent structural results detailing the
postfusion trimeric conformation of DENV type 2 (DENV-2)
and tick-borne encephalitis virus E proteins has prompted a
new model for type II viral fusion (7, 47). In the postfusion
trimer, there is a reorganized E protein domain structure, with
significant exposure of the hydrophobic fusion peptide in DII
The majority of flavivirus-neutralizing antibodies recognize the
structural E protein, although some also bind to the prM/M pro-
* Corresponding author. Mailing address: Division of Infectious Dis-
eases, Department of Medicine, Washington University School of Medi-
cine, Campus Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110. Phone:
† S.S.-P. and S.K.A. contributed equally to the manuscript.
?Published ahead of print on 19 September 2007.
tein (16, 21, 58, 73). Serotype-specific epitopes elicit antibodies
animals by antibodies correlates with neutralizing activity in vitro
(6, 20, 25, 43, 53, 63). Based on epitope mapping data, many
type-specific neutralizing antibodies against individual flaviviruses
localize to DIII (1, 2, 10, 18, 39, 53, 61, 65, 66, 76), whereas
neutralizing monoclonal antibodies (MAbs) that cross-react with
(17, 23, 24, 54, 61, 68). Alteration of specific residues in DIII
results in the loss of binding of neutralizing MAbs (2, 12, 32, 39,
40, 49). Recently, our group, along with others, localized individ-
ual contact residues of a large panel of anti-West Nile virus
(anti-WNV) MAbs and defined a dominant neutralizing epitope
on the lateral ridge of DIII (2, 53, 64). Crystallographic analysis
indicated that a strongly neutralizing, DIII-specific anti-WNV
MAb engaged four discontinuous segments, including the N-ter-
minal linker region (residues 302 to 309) and three strand-con-
to 368), and FG (residues 389 to 391), which together form a
single concave surface patch (51). Comparison of available WNV
sequences revealed nearly complete conservation of the structur-
ally defined epitope. However, sequence analysis of other flavivi-
ruses revealed diversity in the four segments, with notable varia-
tion even between DENV serotypes. Interestingly, other groups
have also identified individual flavivirus-specific neutralizing an-
tibodies that localize to an analogous DIII binding region (32, 74,
75). Based on the coincident mapping of our and other neutral-
izing MAbs, we predicted that this structural epitope, although
specific for individual flaviviruses, would have a dominant role in
neutralization of all flaviviruses (51, 52).
In this study, we mapped the contact residues of a panel of
anti-DENV-2, DIII-specific MAbs with distinct neutralizing po-
tentials. Type-specific MAbs with the strongest neutralizing activ-
ities against DENV-2 localized to an epitope on the lateral ridge
of DIII that was analogous in location to that seen with neutral-
izing WNV MAbs. Subcomplex-specific neutralizing MAbs that
recognized several serotypes of DENV bound an adjacent
epitope centered on the A strand of DIII. In contrast, several
poorly neutralizing MAbs recognized conserved flavivirus resi-
dues within the AB loop that appear to have limited accessibility
on the mature virion. Overall, in contrast to previous studies with
WNV DIII, our data suggest the existence of two structurally
distinct neutralizing epitopes on DIII of DENV-2 E protein, with
a type-specific epitope on the lateral ridge of DIII centered at the
unique FG loop and a subcomplex-specific epitope that binds the
more conserved A strand.
MATERIALS AND METHODS
Cells and viruses. Vero, BHK21-15, and Raji-DC-SIGN-R cells were main-
tained at 37°C in a 5% CO2incubator in Dulbecco’s modified essential medium
or RPMI 1640 supplemented with 10% fetal bovine serum (Omega Scientific
Inc., Tarzana, CA), 1% penicillin G, and 1% streptomycin. The DENV strains
used were 16007 (DENV-1), 16681 (DENV-2), 16652 (DENV-3), and H241
(DENV-4). The viruses were amplified in C6/36 cells according to established
protocols (19). Plaque reduction neutralization titer (PRNT) assays were per-
formed on BHK21-15 cells with individual MAbs and quantitated as previously
MAbs. The MAbs used in this study are presented in Table 1 and were purified
from hybridoma culture supernatants or mouse ascites fluid by using an NAB
protein A spin purification kit (Pierce, Rockford, IL). For sorting experiments,
MAbs were labeled with Alexa Fluor 647 or Alexa Fluor 488, using a MAb
labeling kit (Molecular Probes, Invitrogen, Carlsbad, CA).
Expression of DENV-2 E protein DIII in yeast. The DNA fragment encoding
amino acid residues 294 to 409 (DIII) of the DENV-2 E protein was amplified
from a DENV-2 strain 16681 infectious cDNA clone (35) by PCR, with BamHI
and XhoI sites added at the 5? and 3? ends, respectively. The PCR products were
digested and cloned as downstream fusions to Aga2 and Xpress epitope tag
genes in the yeast surface display vector pYD1 (Invitrogen, Carlsbad, CA), under
the control of an upstream GAL1 promoter. These constructs were transformed
into Saccharomyces cerevisiae strain EBY100 (4, 5), using an S.c. EasyComp
transformation kit (Invitrogen, Carlsbad, CA), to generate yeast that expressed
DENV-2 DIII. Individual yeast colonies were grown to logarithmic phase at 30°C
in tryptophan-free yeast medium containing 2% glucose. Fusion protein expres-
sion was induced on the surface by growing yeast cells for an additional 48 h in
tryptophan-free medium containing 2% galactose at 20°C. Yeast cells were
harvested, washed with phosphate-buffered saline (PBS) supplemented with bo-
vine serum albumin (1 mg/ml), and stained with 50 ?l of diluted MAbs. Purified
antibodies were used at a concentration of 50 ?g/ml, and MAbs from ascites fluid
were used at a 1:100 dilution. After a 30-min incubation on ice, yeast cells were
washed in PBS with bovine serum albumin and then stained with a goat anti-
mouse immunoglobulin G (IgG) secondary antibody conjugated to Alexa Fluor
647 (Molecular Probes, Invitrogen, Carlsbad, CA). After fixation with 1% para-
formaldehyde in PBS, yeast cells were analyzed on a FACSCalibur flow cytom-
eter (Becton Dickinson, Franklin Lakes, NJ).
Library construction and screening. To generate a random mutant library,
DENV-2 DIII was mutated by error-prone PCR, using a GeneMorph II random
mutagenesis kit (Stratagene). The mutant library was ligated into the pYD1
vector and transformed into XL2-Blue ultracompetent cells (Stratagene, La
Jolla, CA). The colonies were pooled and transformed into yeast cells as de-
For each individual MAb, the DENV-2 DIII mutant library was screened
according to a previously described protocol (53). To identify yeasts that had
selectively lost binding to a given MAb, the library was initially stained with this
antibody conjugated to Alexa Fluor 647 for 30 min on ice. To control for the
surface expression of DENV-2 DIII, yeast cells were subsequently stained for 30
min on ice with an Alexa Fluor 488-conjugated oligoclonal antibody that was
derived from a pool of individual MAbs (1F1, 1A1D-2, 6B6-10, 9A3D-8, 13D4-1,
and 5A2-7). After being stained, yeast cells were subjected to flow cytometry, and
the population that was single MAb negative but oligoclonal antibody positive
was identified. After three or four rounds of sorting, yeast cells were plated and
individual colonies were tested for binding to individual MAbs by flow cytometry.
For individual clones that had lost only the desired MAb binding epitope, the
pYD1-DV2 DIII plasmid was recovered using a Zymoprep yeast miniprep kit
(Zymo Research, Orange, CA). The plasmid was transformed into XL1-Blue
competent cells (Stratagene, La Jolla, CA), purified using a QIAprep spin mini-
prep kit (QIAGEN), and sequenced. In some cases, DENV-2 DIII variants with
multiple mutations were isolated. To determine which mutation conferred the
phenotype, single independent mutations were engineered by site-directed mu-
tagenesis, using a QuikChange II mutagenesis kit (Stratagene, La Jolla, CA), or
by using splice-overlap PCR (45).
MAb staining of DENV-infected cells. Raji-DC-SIGN-R cells were infected
with DENV-1, DENV-2, DENV-3, or DENV-4 at a multiplicity of infection of
TABLE 1. Characteristics of DIII-specific MAbs used in this study
aDetermined by PRNT assay on BHK21 cells with 102PFU of DENV-2.
VOL. 81, 2007MAPPING OF DENV-2 NEUTRALIZING MAbs ON DIII 12817
0.5 or 1. Depending on the strain, cells were harvested at 72 or 96 h, washed three
times in PBS, and fixed in PBS with 1% paraformaldehyde. Cells were then
washed twice in PBS and permeabilized in Hanks’ balanced salt solution (Cell-
gro, Herndon, VA) containing 10 mM HEPES (pH 7.3), 0.1% saponin (Sigma,
St. Louis, MO), and 0.02% NaN3(HHSN). MAbs were bound to permeabilized
virus-infected cells for 30 min on ice, washed three times in HHSN, and resus-
pended in 50 ?l of a 1/500 dilution of Alexa Fluor-conjugated anti-mouse IgG
(Molecular Probes, Invitrogen, Carlsbad, CA). After 15 min, cells were again
washed in HHSN three times, fixed in PBS with 1% paraformaldehyde, and
processed by flow cytometry.
Mapping of mutations onto the DENV-2 DIII crystal structure and virion.
Figures were prepared using the atomic coordinates of DENV-2 E (Research
Collaboratory for Structural Bioinformatics [RCSB] accession number 1OAN)
and the WNV E DIII/E16 Fab complex (RCSB accession number 1ZTX), using
the programs Ribbons (9), MOLEMAN2 (36), Insight II (Accelrys, San Diego,
CA), and PyMol (http://www.pymol.org). The representations of the DENV-2
virion were generated using the atomic coordinates and transformation matrices
found in RCSB entry 1THD.
Strongly neutralizing MAbs against WNV localize to an
epitope (see Fig. 2A) composed of the loops from three
?-strands and an N-terminal region on the lateral ridge of DIII
(2, 14, 51, 53, 64, 74). Sequence analysis indicated that this
neutralizing epitope is highly conserved among WNV strains
but divergent from those of other flaviviruses (38, 51). To test
whether the analogous epitope on DIII of DENV-2 elicited
strongly neutralizing antibodies, we screened a panel of 40
MAbs obtained from colleagues for immunoreactivity with
DENV-2 strain 16681. Twenty-one of 40 MAbs exhibited sig-
nificant reactivity with DENV-2-infected cells or purified re-
combinant soluble DENV-2 E protein derived from insect cells
(data not shown). Of the MAbs that recognized DENV-2-
infected cells, 14 bound strongly to yeast cells displaying
DENV-2 DIII (data not shown). Of these, four had moderate
(50% PRNT [PRNT50], 1 to 100 ?g/ml) and six had strong
(PRNT50?1 ?g/ml) neutralizing activities against strain 16681
in a standard PRNT assay (Table 1). Four others had weak or
no appreciable inhibitory activity (PRNT50, ?100 ?g/ml). No-
tably, five of the six strongly neutralizing MAbs were type
specific and showed no significant cross-reactivity with DENV-
1-, DENV-3-, or DENV-4-infected cells (Table 2).
To map the amino acid contact residues of the DIII-specific
DENV-2 MAbs with distinct neutralizing properties, we ap-
plied a high-throughput strategy that previously mapped 167
WNV E protein-specific MAbs (53, 54). Error-prone PCR
mutagenesis introduced random point mutations within DIII
of DENV-2 E. A library of ?3.5 ? 105variants was pooled and
used to create a mutant yeast expression library. Individual
screens were performed to identify DIII mutants that lost
binding selectively to strongly neutralizing MAbs. To eliminate
mutants that abolished surface expression of DIII, yeast cells
were stained sequentially with an Alexa Fluor 647-conjugated
individual MAb and an Alexa Fluor 488-conjugated oligo-
clonal antibody derived from a pool of individual MAbs. After
several rounds of cell sorting, yeast cells that selectively lost
expression of an individual MAb epitope but retained expres-
sion of DIII on the surface were identified. Multiple indepen-
dent yeast clones that selectively lost binding of individual
MAbs were subjected to plasmid recovery and sequencing.
Subsequently, mutant DIII expressed on the yeast surface was
tested for MAb reactivity against the remainder of the panel of
MAbs by flow cytometry (Fig. 1 and data not shown).
From each screen, we recovered 6 to 20 independent mutants
that lost binding for an individual MAb. After sequencing them,
we discovered that some of these mutants contained multiple
mutations within the DIII region. In such cases, single mutations
were engineered separately by site-directed mutagenesis to iden-
tify the change that caused the phenotype. Type-specific MAbs
that localized to DIII and neutralized DENV-2 strongly (1F1,
3H5-1, 6B6-10, 9A3D-8, and 9F16) showed markedly reduced
binding (?80% reduction) when residues G304 (five of five
MAbs), E383 (three of five MAbs), and P384 (four of five MAbs)
were altered (Fig. 1 and Table 3). Similarly, type-specific MAbs
that neutralized moderately (PRNT50of 2 to 5 ?g/ml; M8051122,
9F11, and 2Q1899) also showed decreased binding when these
three residues were changed. G304, E383, and P384 are located
on adjacent loops and form a contiguous patch on the solvent-
exposed surface at the lateral ridge of DIII (Fig. 2). As observed
for DIII-specific neutralizing MAbs against WNV (53), the type-
specific neutralizing DENV-2 MAbs bound overlapping epitopes.
TABLE 2. Binding of MAbs to Raji-DC-SIGN-R cells infected with DENV-1, -2, -3, and -4
Binding to virusa
DENV-1 16007DENV-2 16681DENV-3 16652DENV-4 H241
a???, strong binding (40 to 100% compared to control) to infected cells; ?, weak binding (15 to 40% compared to control) to infected cells; ?, no appreciable
12818SUKUPOLVI-PETTY ET AL.J. VIROL.
Consistent with this, the following additional point mutations
caused significant loss of binding of individual type-specific neu-
tralizing MAbs tested: T303Y (1F1), K307E (9A3D-8), E327R
(9A3D-8), D329R (6B6-10), G330D (1F1 and 6B6-10), and
S331Y (6B6-10). Mutation of G304, which appears to comprise
part of a type-specific neutralizing epitope on DENV-2 DIII, also
affected binding of both subcomplex-specific MAbs (9D12 and
1A1D-2) and one non- or weakly neutralizing MAb (E114). Be-
cause binding of several MAbs of different classes was affected by
the structure of this mutant DIII. Against this, we did observe
relatively wild-type levels of binding of two other nonneutralizing
MAbs (13D4-1 and E111) to this mutant.
In our panel of MAbs, we also identified two neutralizing
MAbs (9D12 and 1A1D-2) that reacted with additional DENV
serotypes. These subcomplex-specific MAbs showed moderate
and strong inhibitory activities against DENV-2 infection, re-
spectively (Tables 1 and 2). These MAbs, however, bound DIII
somewhat distinctly. 1A1D-2, which binds DENV-1, DENV-2,
and DENV-3, retained relatively normal binding with E383G
and P384N mutations but showed markedly reduced binding
with mutations in residues K305, K307, and K310. In compar-
ison, 9D12, which binds DENV-2 and DENV-4 and weakly
binds DENV-1, retained binding with the E383G and P384A
mutations but had markedly reduced binding with K305E,
K307E, K310E, and P384N mutations (Table 3).
Also in our panel were four MAbs (5A2-7, 13D4-1, E111,
and E114) that had little or no neutralizing activity (Table
1). This group of MAbs recognized all four serotypes of
DENV (Table 2). Two of them, E111 and E114, were cross-
reactive and also recognized yeast expressing WNV DIII
(data not shown). Independent yeast sorting for loss-of-
binding mutations was performed with three of the four
MAbs. Several mutations that specifically reduced binding
of the poorly neutralizing MAbs but had little effect on most
strongly neutralizing MAbs were identified. For example,
H317Y mutation specifically reduced binding of the 5A2-7,
13D4-1, and E111 MAbs (Table 3), and a DIII variant with
two mutations (T315G and S331Y) also diminished binding
of these MAbs but did not affect any neutralizing MAbs,
with the exception of 6B6-10. Interestingly, the T315G sin-
gle mutant only modestly decreased 5A2-7 (71% reduction)
binding, and the S331Y single mutant had no effect on
binding of 5A2-7, 13D4-1, or E111.
To visualize spatially the different recognition patterns of
MAbs that strongly, moderately, and weakly neutralized
DENV-2 infection, we docked the loss-of-binding mutations
(?20% of wild-type binding) defined by the yeast assay onto
the existing crystallographic structure of DENV-2 DIII,
the prefusion DENV-2 E protein dimer structure (46), and the
pseudoatomic model of the mature DENV-2 virion (37). The
DIII structures were compared to the previously defined crys-
tallographic epitope (16 contact residues) of E16 on the lateral
ridge of DIII of WNV (51) (Fig. 2A and B). Type-specific
MAbs with the strongest neutralizing activities (3H5-1, 6B6-10,
and 1F1) localized to amino acids on the analogous lateral
ridge of DIII (Fig. 2D, E, and F). Nonetheless, the yeast
mapping did suggest some subtle differences. Whereas the
lateral ridge epitope of the strongly neutralizing anti-WNV
MAb E16 was centered on the N-terminal region and the BC
loop (K307, T330, and T332 of WNV), strongly neutralizing
type-specific anti-DENV-2 MAbs localized more to the FG
loop (E383 and P384 of DENV-2). The E383 and P384 resi-
dues in the FG loop are highly conserved among DENV-2
isolates but are not present in DENV-1, DENV-3, or DENV-4
strains (Fig. 3). The epitope of type-specific neutralizing MAbs
against DENV-2 followed the same exposure pattern on the
virion as that previously identified for E16 (34, 51): two of the
FIG. 1. Flow cytometry histograms of loss-of-function DIII variants (G304Y, K307N, D329G, and P384N) selected by yeast surface display after
being sorted with MAbs. Representative histograms are shown for MAbs 1F1, 3H5-1, and 13D4-1 with wild-type DIII (WT) and each of the DIII
mutants. Data shown are representative of three independent experiments.
VOL. 81, 2007 MAPPING OF DENV-2 NEUTRALIZING MAbs ON DIII12819
TABLE 3. Summary of MAb binding to DENV-2 DIII mutants expressed on the surfaces of yeast cells
MAb binding to mutanta
T303Y G304Y K305E K307N K307Q K307I K307E K310E T315G
H317Y R323E E327R D329G D329R G330D S331Y E338D T359I E383G P384A P384N N390H N390Y
aValues shown were obtained by dividing the total fluorescence product (percent positive population ? mean linear fluorescence intensity) for a mutant for each individual antibody by the total fluorescence product
for wild-type DIII. Values in bold indicate reductions in MAb binding of ?80% for a given mutation. The results are averages for three to five independent experiments for each mutant and each antibody. Since the
G304Y mutant had slightly less surface expression on yeast cells, the values for this mutant only were normalized to the strongest binding antibody for that mutant ? 100. ND, not determined.
12820SUKUPOLVI-PETTY ET AL. J. VIROL.
three DIIIs per icosahedral asymmetric unit were exposed,
with steric hindrance noted at the fivefold clustered DIII
Subcomplex-specific MAbs (1A1D-2 and 9D12) that strongly
and moderately neutralized DENV-2 infection recognized a
flanking epitope (Fig. 4A and B). This epitope was centered on
amino acids K305, K307, and K310 on the A strand. Consistent
with the limited cross-reactivity of these subcomplex-specific
MAbs, K310 is completely conserved among all four DENV se-
rotypes, whereas K305 and K307 are conserved in DENV-4 and
DENV-1 strains, respectively (Fig. 3). Although the 1A1D-2 and
environments on the mature virion (Fig. 4C and data not shown),
some differences with the type-specific lateral ridge epitope were
apparent, as follows: the 9D12 epitope appears predominantly
exposed on the fivefold clustered DIII, and thus, one could spec-
ulate that a full complement of 180 Fabs could bind DENV-2 at
Several of the poorly neutralizing, cross-reactive MAbs
(5A2-7, 13D4-1, and E111) mapped to an amino acid residue
(e.g., H317) that was localized to the back side of DIII in the
AB loop (Fig. 5A and B). Consistent with the cross-reactive
nature of these MAbs, the entire AB loop sequence (E314,
T315, Q316, H317, G318, and T319) is completely conserved
among all four DENV serotypes. Moreover, the H317, G318,
and T319 residues are present in virtually all WNV isolates and
the H317 and T319 amino acids are conserved among flavivi-
ruses (Fig. 3 and data not shown). Structurally, the AB loop
has limited exposure on the surface of the E protein dimer and
faces inward toward the nucleocapsid in all three symmetry
environments of the mature virion (Fig. 5C and Fig. 6).
The goal of this study was to characterize epitopes on DIII
recognized by potent neutralizing antibodies against DENV-2.
FIG. 2. DIII lateral ridge antibody epitope. The structures of WNV and DENV-2 DIIIs are shown, with identification of binding sites of type-specific
neutralizing antibodies. (A) Structure of WNV E16 neutralizing epitope determined by X-ray crystallography (16 residues in blue) or by using yeast display
mapping (4 residues in orange). (B) Structure of DENV-2 DIII, with the corresponding 16 amino acids of the WNV E16 neutralizing antibody epitope
highlighted. (C) Yeast display epitope residues (red) for the 1F1 MAb were mapped onto the pseudoatomic model of the mature DENV-2 virion (37). Virions
are depicted as 2.0-Å-radius C-? atoms and are colored according to their E protein symmetry relationships, i.e., twofold (cyan), threefold (green), or fivefold
(yellow) symmetry. (D to F) Structure of DENV DIII, with amino acid residues that significantly affect binding of type-specific neutralizing MAbs 3H5-1 (D),
6B6-10 (E), and 1F1 (F) marked in orange. The DIII disulfide bond is depicted in yellow.
VOL. 81, 2007MAPPING OF DENV-2 NEUTRALIZING MAbs ON DIII 12821
Previous studies had established a dominant type-specific
epitope for eliciting protective antibodies in vitro and in vivo
against the related flavivirus WNV. Here we tested a panel of
MAbs against DIII of the DENV-2 E protein. Type-specific
strongly neutralizing MAbs mapped to an analogous epitope
centered on the FG loop of the lateral ridge region on DIII of
DENV-2. Subcomplex-specific strongly neutralizing MAbs lo-
calized to a flanking epitope that was centered on three lysine
residues in the A strand of DIII.
Extensive MAb competition binding studies have been per-
formed by several groups to identify distinct antigenic and
functional determinants on DENV-2 (18, 22, 29, 30, 61). Po-
tently inhibitory type-specific MAbs were localized to domain
B, which is now called DIII, based on structural analysis of the
domain organization of flavivirus E proteins (46, 60). None-
theless, amino acid contact residues of few neutralizing MAbs
that react with DENV-2 have been established. For these
(3H5-1, 4E11, G8D11, and 4G2), precise mapping data were
obtained by analyzing neutralization escape mutants (39) and
by differential recognition of chimeric DENV variants (32),
site-specific DENV-2 mutants (17, 67), and E protein peptide
sequences (44, 70, 72). We used a forward genetic strategy,
error-prone PCR mutagenesis of DIII of DENV-2 E protein,
and expression on yeast cells to map antibody contact residues
in a nonbiased manner. By having a panel of DIII MAbs with
differing neutralization potentials, we minimized the possibility
that mutations would grossly affect folding. The validity of the
yeast approach for identifying critical contact residues was
FIG. 3. DIII amino acid sequence alignment. The sequence and secondary structure of DIII from the DENV-2 (strain 16681) E protein are
aligned with those for DENV-1 (strain 16007), DENV-3 (strain 16652), DENV-4 (strain 1036), and WNV (New York 1999). The secondary
structure of DENV-2 E DIII residues 294 to 395 (RCSB entry 1OAN) was predicted by DSSP (33). The results of yeast surface display epitope
mapping are highlighted, with DENV-2 residues recognized primarily by type-specific MAbs colored magenta, subcomplex-specific residues
colored green, and cross-reactive residues colored orange. The residues contacted by E16 on WNV DIII, as determined by crystallography, are
colored blue (51), with black asterisks denoting residues identified by yeast display (53). Colored asterisks denote DENV-2 residues that are
recognized by multiple classes of antibodies. For example, G304Y mutation resulted in a loss of binding of all type- and subcomplex-specific MAbs
and a single cross-reactive MAb.
FIG. 4. DIII A-strand epitope. The structure of DENV-2 DIII is shown, with identification of binding sites of subcomplex-specific neutralizing
antibodies. (A and B) Structure of DENV DIII, with amino acid residues that significantly affect binding of subcomplex-specific neutralizing MAbs
1A1D-2 (A) and 9D12 (B) marked in orange. (C) Yeast display epitope residues (red) for the 9D12 MAb were mapped onto the pseudoatomic
model of the mature DENV-2 virion. Virions are depicted as described in the legend to Fig. 2.
12822 SUKUPOLVI-PETTY ET AL.J. VIROL.
confirmed by X-ray crystallographic studies that resolved the
structural interface between DIII and a neutralizing anti-WNV
Fab fragment (51). Of the DENV-2 MAbs that have been
reported to contact specific amino acid residues in DIII, only
the type-specific MAb 3H5-1 was available for our analysis.
Prior fine mapping studies suggested that 3H5-1 recognized
either a Glu-Pro-Gly motif centered at amino acids 383, 384,
and 385 (32) or a linear peptide encompassing amino acids 386
to 397 (32, 72). Our yeast mapping experiments confirmed an
essential role for residues E383 and P384 but also suggested an
additional important contact residue (G304) located on an
DIII of the E protein adopts an immunoglobulin-like fold
(46, 60) that is significantly exposed on the surface of the
mature virion (50, 78). The lateral ridge epitope on DIII was
previously defined by X-ray crystallographic and nuclear mag-
FIG. 5. DIII AB loop epitope. The structure of DENV-2 DIII is shown, with identification of binding sites of poorly neutralizing antibodies.
(A and B) Structure of DENV DIII, with amino acid residues that significantly affect binding of the poorly neutralizing MAbs E111 (A) and 13D4-1
(B) marked in orange. (C) Yeast display epitope residues (red) for the 13D4-1 MAb were mapped onto the pseudoatomic model of the mature
DENV-2 virion. Virions are depicted as described in the legend to Fig. 2. Note that the 13D4-1 epitope is poorly accessible on the virion compared
to the 1F1 (Fig. 2C) and 9D12 (Fig. 4C) epitopes.
FIG. 6. Mapping of MAb epitopes onto the DENV-2 E protein dimer. The yeast display epitope residues (in red) for the type-specific 1F1 (A),
subcomplex-specific 9D12 (B), and cross-reactive 13D4-1 (C) MAbs were rendered on the crystal structure of the DENV-2 E protein dimer (46)
and are shown in side view, with the bottom side facing the viral lipid membrane.
VOL. 81, 2007 MAPPING OF DENV-2 NEUTRALIZING MAbs ON DIII12823
netic resonance studies of Fab-DIII complexes of WNV and
Japanese encephalitis virus and encompasses four discontinu-
ous loops (51, 74, 75). In our study, we found six different
strongly neutralizing MAbs against DENV-2 that localized to
two overlapping structural epitopes on the lateral ridge and
adjacent A strand of DIII.
For flaviviruses, virus type-specific epitopes generally elicit
the most potent neutralizing antibodies (2, 28, 42, 53, 56, 63,
64). Of the DIII-specific MAbs against DENV-2 in our panel,
in general, the ones with the strongest neutralizing activities
were type specific and localized to the lateral ridge of DIII
centered at the FG loop, near residues E383 and P384. None-
theless, some of the type-specific neutralizing MAbs inhibited
virus infectivity less strongly, although they recognized similar
residues. Although further biophysical studies are needed, the
affinities of binding of MAbs for a given DENV-2 DIII epitope
may correlate with relative occupancy and may predict the
strength of neutralization. Such a result was observed with less
strongly neutralizing MAbs that recognized the lateral ridge
epitope of DIII of WNV (57).
The two subcomplex-specific (1A1D-2 [PRNT50, 0.3 ?g/ml]
and 9D12 [PRNT50, 2 ?g/ml]) MAbs that we tested had strong
and moderate neutralizing activities, respectively, consistent
with prior studies with the group-specific MAb 4E11 (PRNT50
values of 0.3 to 2.4 ?g/ml for DENV-1 to DENV-4) (71).
1A1D-2 and 9D12 bind a more conserved epitope among
DENV serotypes that partially overlaps with the type-specific
lateral ridge neutralizing epitope but is centered on the A
strand at residues K305, K307, and K310. Notably, several of
these amino acids were recently identified as contact residues
for the MAb 4E11, which neutralizes all four DENV serotypes
(41, 70). Such broadly neutralizing subcomplex- or group-spe-
cific MAbs may have potential for development as antibody-
based therapeutics against all serotypes of DENV.
An epitope map analysis of the different DENV-2 MAbs at
the amino acid sequence level begins to explain their serotype-
specific properties. Type-specific neutralizing MAbs against
DENV-2 are centered on amino acids E383 and P384 in the
FG loop, which are conserved among DENV-2 isolates but
divergent in all other DENV serotypes. Subcomplex-specific
neutralizing MAbs that bind some but not all DENV serotypes
recognize a distinct epitope centered on three A-strand lysines
of DIII. K310 is completely conserved among all four DENV
serotypes, whereas K305 and K307 are conserved in DENV-4
and DENV-1 strains, respectively. The distinct binding speci-
ficities of 9D12 and 1A1D-2 for other DENV serotypes likely
reflect differences in interactions with specific lysines on the A
strand and the divergence of other additional contact residues
in the FG loop and the G strand. Cross-reactive poorly neu-
tralizing MAbs localized to the AB loop on DIII. Their lack of
neutralizing activity is probably explained by the limited sur-
face exposure of the AB loop. As seen for the DIII-specific
poorly neutralizing anti-WNV MAb E9, a relative lack of ac-
cessibility directly affects the stoichiometry of binding, such
that a threshold for antibody neutralization is rarely reached
(57). The cross-reactivity of these AB loop MAbs with all
DENV serotypes and distantly related flaviviruses is reason-
ably explained by sequence conservation: amino acids 314 to
319 are completely conserved among all four DENV serotypes,
and residues 317 and 319 (which include the critical H317
amino acid) are conserved in virtually all flaviviruses. This
analysis has implications for the development of novel DIII
epitope-based diagnostic reagents for polyclonal antibodies, as
mutation of AB loop sequences could abolish serotype and
flavivirus nonneutralizing cross-reactive epitopes, making
DIII-based immunologic assays more predictive of serotype-
specific neutralizing antibodies.
Although the sequence of the lateral ridge epitope of DIII is
variable among flaviviruses, the majority of mutations that
abolish binding of virus-specific strongly neutralizing antibod-
ies map there (2, 10, 12, 18, 53, 61, 63, 64, 67, 75), suggesting
the existence of a type-specific neutralizing epitope for flavivi-
ruses on DIII. It is important, however, that these epitope
maps were derived using murine MAbs. Whether the human
antibody response recognizes the same or different epitopes
has not yet been determined in detail. Only 8% (4 of 51
antibodies) and 0% (0 of 11 antibodies) of human single-chain
antibodies that were isolated from phage display libraries of
WNV-infected or naı ¨ve patients reacted with DIII (26, 69).
These results suggest that the human antibody repertoire
against flaviviruses may actually be directed away from these
DIII neutralizing epitopes and toward the inherently less neu-
tralizing immunodominant epitopes on DI and DII (55). Since
our results suggest that strongly neutralizing antibodies against
DIII of DENV-2 map to the lateral ridge and A-strand
epitopes, flavivirus DIII-based vaccines (13, 15, 31) that inten-
tionally skew the humoral response to these epitopes and away
from the cross-reactive, poorly accessible epitope in the AB
loop could elicit a greater protective response.
We thank C. Nelson for help with structural analysis of DENV E
proteins, members of our laboratories for critical reviews of the manu-
script, and R. Putnak for providing several of the MAbs used in this
This work was supported by the Pediatric Dengue Vaccine Initiative
(M.S.D., D.H.F., J.T.R., J.J.S., and A.D.B.) and by NIH grants
AI061373 (M.S.D.) and U54 AI057160 (Midwest Regional Center of
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