A Novel Neutralizing Antibody Specific to the

DE Loop of VP1 Can Inhibit EV-D68

This information is current as
of February 1, 2021.
Infection in Mice
Huiwen Zheng, Jingjing Wang, Bingxiang Li, Lei Guo,
Heng Li, Jie Song, Zening Yang, Hongzhe Li, Haitao Fan,
Xing Huang, Haiting Long, Chen Cheng, Manman Chu,
Zhanlong He, Wenhai Yu, Jiaqi Li, You Gao, Ruotong Ning,
Nan Li, Jinxi Yang, Qiongwen Wu, Haijing Shi, Ming Sun
and Longding Liu

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

J Immunol 2018; 201:2557-2569; Prepublished online 3
October 2018;
doi: 10.4049/jimmunol.1800655
http://www.jimmunol.org/content/201/9/2557

References This article cites 54 articles, 12 of which you can access for free at:
http://www.jimmunol.org/content/201/9/2557.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision
• No Triage! Every submission reviewed by practicing scientists
• Fast Publication! 4 weeks from acceptance to publication
*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription
Permissions Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
Errata An erratum has been published regarding this article. Please see next page
or:
/content/202/6/1905.full.pdf

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2018 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
 The Journal of Immunology

A Novel Neutralizing Antibody Specific to the DE Loop of

VP1 Can Inhibit EV-D68 Infection in Mice

Huiwen Zheng,1 Jingjing Wang,1 Bingxiang Li,1 Lei Guo, Heng Li, Jie Song,
Zening Yang, Hongzhe Li, Haitao Fan, Xing Huang, Haiting Long, Chen Cheng,
Manman Chu, Zhanlong He, Wenhai Yu, Jiaqi Li, You Gao, Ruotong Ning,
Nan Li, Jinxi Yang, Qiongwen Wu, Haijing Shi, Ming Sun, and Longding Liu
Enterovirus D68 (EV-D68) belongs to the picornavirus family and was first isolated in CA, USA, in 1962. EV-D68 can cause severe
cranial nerve system damage such as flaccid paralysis and acute respiratory diseases such as pneumonia. There are currently no
efficient therapeutic methods or effective prophylactics. In this study, we isolated the mAb A6-1 from an EV-D68–infected rhesus
macaque (Macaca mulatta) and found that the Ab provided effective protection in EV-D68 intranasally infected suckling mice. We
observed that A6-1 bound to the DE loop of EV-D68 VP1 and interfered with the interaction between the EV-D68 virus and

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

a2,6-linked sialic acids of the host cell. The production of A6-1 and its Ab properties present a bridging study for EV-D68 vaccine
design and provide a tool for analyzing the process by which Abs can inhibit EV-D68 infection. The Journal of Immunology,
2018, 201: 2557–2569.

E
nterovirus D68 (EV-D68) is a major causative agent of
mild to severe upper and lower respiratory illnesses and is
prevalent in North America, Europe, and Asia in recent
years (1–3). Patients infected with EV-D68 exhibit fever, sneezing,
rhinorrhea, cough, sore throat, pneumonia, asthma exacerbation,
and acute flaccid myelitis, with the possibility of death (2, 4–7).
No prophylactic vaccine or therapeutic drug against EV-D68 in-
fection is yet available. Potent Abs represent a new generation of
antiviral agents for the prophylaxis and treatment of viral infection
(8, 9). Thus, the induction of an Ab response capable of neutral-
izing EV-D68 isolates is urgently needed to address critical clin-
ical conditions.
Many new mAbs have been successfully isolated and charac-
terized by the microneutralization screening of single B cell sorting
from infected patients (10, 11). As a widely used type of animal
model, nonhuman primates such as Chinese rhesus macaques in-
fected by a novel pathogen provide valuable information regard-
ing the immune response to infection (12–16). The identification
of the functional antisera elicited by infection, including bind-
ing capabilities and neutralizing titers, is vitally important for
the study of novel pathogens (17, 18). Moreover, comprehensive
assessment focusing on the quality, features, and antiviral activity
of neutralizing Abs might be conducted to fully define the mAb
candidates induced by an infection (19, 20).
EV-D68 is a nonenveloped, single positive-strand RNA virus
with an icosahedron structure. There is a “canyon” region on the
EV-D68 surface that contains the sialic acid receptor binding
sites (21, 22). In addition, the VP1 protein in this canyon is an
important structural protein for EV-D68 virus–dominant Ags that
has also been used to classify virus genotypes. Despite the va-
riety of strains of EV-D68, the strains detected recently have
similar VP1 sequences as long as they belong to the same genetic
lineage.
In this study, we report the isolation of a class of VP1-specific
mAbs from an EV-D68–infected rhesus macaque and their neu-
tralization activities. A6-1, the most potent of the isolated Abs,
exhibited many promising characteristics including a specific
Ag–binding capacity, affinity, and specific reactivity. In addition,
biochemical binding studies and the characterization of the epi-
topes recognized by A6-1 demonstrate that its potency is mediated
by its unique mode of recognition of a conserved site of vulnerability
within the VP1-DE loop located in the canyon region by sequentially

Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking
Union Medical College, Kunming 650118, China; and Key Laboratory of Systemic
Innovative Research on Virus Vaccine, Chinese Academy of Medical Sciences,
Kunming 650118, China
1
H.Z., J.W., and B.L. contributed equally to this work.
ORCIDs: 0000-0002-3852-258X (J.Y.); 0000-0002-6430-5677 (L.L.).
Received for publication May 9, 2018. Accepted for publication August 30, 2018.
This work was supported by the Chinese Academy of Medical Sciences Innovation
Fund for Medical Sciences (2016-I2M-1-014), the Yunnan Provincial Innovation
Team of China (2016HC009), and the National Nature Science Foundation of China
(31570900 and 81373142). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
L.L. and M.S. conceived, designed, and supervised the project. H.Z., J.W., and B.L.
performed single B cell sort, PCR, and Ab expression and activity tests. L.G., J.S.,
Heng Li, Z.H., and W.Y. performed the animal experiments and cell biology exper-
iments; Z.Y., Hongzhe Li, H.F., X.H., J.L., R.N., and H.S. captured and analyzed the
images. C.C., H. Long, M.C., and Y.G. carried out the cell culture and virus isolation.
N.L., J.Y., and Q.W. contributed reagents/materials/analysis tools; L.L., M.S., H.Z.,
B.L., and J.W. analyzed all data and wrote the paper.
The sequences presented in this article have been submitted to the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) under accession num-
ber MG991260.
Address correspondence and reprint requests to Prof. Longding Liu and Prof. Ming
Sun, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking
Union Medical College, 935 Jiaoling Road, Kunming 650118, Yunnan, China. E-mail
addresses: longdingl@gmail.com (L.L.) and sunming@imbcams.com.cn (M.S.)
Abbreviations used in this article: CAMS, Chinese Academy of Medical Sciences;
CCID50, cell culture infectious dose; CDRH, complementarity-determining region;
CPE, cytopathic effect; dpi, day postinfection; EV-D68, Enterovirus D68; FLIM,
fluorescence lifetime imaging; FRET, fluorescence resonance energy transfer;
IACUC, Institutional Animal Care and Use Committee; IMB, Institute of Medical
Biology; KM, Kunming; MPI, maximum percent inhibition; MTS, 3-(4,5-dimethyl-
thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBST,
0.1% Tween 20 in PBS; qRT-PCR, quantitative real-time RT-PCR; RT, reverse tran-
scription; STED, stimulated emission depletion; VH, H chain variable gene; VL, L
chain variable gene.
Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800655
2558 A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION
blocking the canyon region from binding with a2,6-linked sialic
acids on the cellular surface through steric hindrance.
A6-1 protected suckling mice against intranasal infection by the
EV-D68 virus. Because A6-1 binds to the DE loop of the EV-D68
VP1 protein, epitopes in this region may be promising candidates
for vaccine design and may be the potential targets for inhibiting
viral infection.
Materials and Methods
Virus and cells
The EV-D68 Kunming (KM) strain (GenBank accession number: MG991260,
https://www.ncbi.nlm.nih.gov/) and the EV-D68 Fermon strain (GenBank
accession number: AY426531.1, https://www.ncbi.nlm.nih.gov/) are pre-
served by the Institute of Medical Biology (IMB), Chinese Academy of
Medical Sciences (CAMS). The CA16 G20 strain and the EV71 FY23 strain
are also preserved by the IMB, CAMS. The virus titer was 6.5 log cell
culture infectious doses (CCID50) per milliliter and was diluted to a suitable
concentration for the subsequent experiments. Vero and HeLa cells were
acquired from the American Type Culture Collection.
Ags and peptides
A series of 60 peptides spanning the entire EV-D68 KM VP1 region of
EV-D68 were synthesized. Each peptide contained 15 aa residues and had
10 of the same amino acid residues as each adjacent peptide. Other 15-aa
peptides of the Fermon strain, which is different from the KM strain in the
VP1 gene sequence, were synthesized. In addition, nine peptides covering
the 126–135 aa of the VP1 protein were synthesized, each with a single
alanine mutation replacement.
Fluorescence labeling of the EV-D68 VP1 protein or A6-1 mAb
We labeled the EV-D68 VP1 protein or A6-1 mAb according to the
Lightning-Link Fluorescein Conjugation Kit instructions (Innova Biosci-
ences, Baraham, Cambridge, U.K.). Before we added purified VP1 protein
(1 mg/ml) to the Lightning-Link mixture, we added 1 ml of LL-modifier
reagent for each 10 ml of VP1 protein to be labeled; then we mixed gently.
We pipetted the VP1 sample (with added LL-modifier) directly onto the
Lightning-Link mixture. We resuspended gently by withdrawing and
redispensing the liquid once or twice using a pipette. After incubating the
mixture at room temperature (20–25˚C) in the dark for 3 h (or more), we
added 1 ml of LL-quencher FD reagent for every 10 ml of VP1 protein
used. After 30 min, the conjugate could then be used.
Animal ethics and the rhesus monkey infection experiment
All animal experiments maintained the replacement, refinement, and re-
duction ethical principles. The animal experiments were approved by the
Institutional Animal Care and Use Committee (IACUC) of the IMB, CAMS.
Two healthy 6-mo-old rhesus monkeys (Macaca mulatta; identifier 15247
weight of 1.15 kg is female, identifier 15249 weight of 1.2 kg is male) were
administered an EV-D68 virus suspension with 4.5 log CCID50 titer via
nasal delivery. Each monkey was kept in a single cage and fed according to
IACUC guidelines. The animal keepers recorded the weight and body
temperature of the monkeys daily and collected blood, nasal swabs, and
fecal specimens from both monkeys at 1, 3, 5, 7, 9, 11, 14, and 28 d
postinfection (dpi). The anti–EV-D68 Ab in both monkeys was confirmed
as negative before the study was initiated.
Quantitative RT-PCR amplification
Total RNA was extracted from different cell cultures, fresh tissue ho-
mogenates, or nasal washes from the experimental animals using the
TRNzol-A+ Reagent Mini Kit (Tiangen Biotech, Beijing, China) according
to the manufacturer’s instructions. The EV-D68 RNA viral loads were
determined using a one-step quantitative real-time RT-PCR (qRT-PCR)
assay as previously described (23).
Single-cell sorting of rhesus macaque Ab-secreting cells by
flow cytometry
For cell surface staining, fresh PBMCs were resuspended in 100 ml of FACS
staining buffer (3% BSA in PBS) containing Abs against CD20, CD27, and
VP1/FITC-labeled Ags and incubated for 30 min at room temperature in
the dark. The stained cells were analyzed and sorted by a BD FACSJazz
cell sorter (BD Biosciences) into 96-well plates containing 20 ml of cell
lysis buffer according to the gating strategy. The CD27+ cells were gated
from CD20+ populations that excluded CD3+ cells. Then, the EV-D68 VP1
Ag–specific memory B cells were defined as CD27+VP1+. All flow
cytometry data were analyzed by FlowJo V10 software (FlowJo, Ashland,
OR). Row H of the 96-well plate was used as a blank control and contained
only lysis buffer from the Takara One Step cDNA Synthesis Kit (Takara
Bio, Japan). The sorted target cells were stored at 280˚C.
RT-PCR and nested PCR
The sequences of the monkey Ig H chain variable gene (VH) and L chain
variable gene (VL) chains were amplified by RT-PCR and nested PCR
using the methods and primers in Table I (24). In brief, Ig VH and VL were
amplified in 96-well PCR plates containing sorted single B cells. The first-
strand cDNA was synthesized according to the PrimeScript II 1st Strand
cDNA Synthesis Kit (Takara Bio), and Ig H chain, Ig lambda chain, and
Ig kappa chain were amplified by two rounds of PCR under the following
conditions.
H chain. The first round of H chain PCR contained 5 ml of the reverse
transcription (RT) reaction, 16 ml of PrimeSTAR Max Premix (23), 1 ml
of each forward primer, and 1 ml of each reverse primer. The second round
of H chain PCR contained 5 ml of the first-round reaction, 19 ml of
PrimeSTAR Max Premix (23), 1 ml of each forward primer, and 1 ml of each
reverse primer. The first-round procedure was as follows: 98˚C for 1 min;
50 cycles of 98˚C for 10 s, 55˚C for 5 s, and 72˚C for 5 s; and finally, 72˚C for
Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021
5 min. The second-round procedure was as follows: 98˚C for 1 min; 50 cycles
of 98˚C for 10 s, 60˚C for 5 s, and 72˚C for 5 s; and finally, 72˚C for 5 min.
L chain l. The first round of L chain l PCR contained 6.5 ml of the RT
reaction, 15 ml of PrimeSTAR Max Premix (23), and 0.8 ml of each
forward primer. The second round of L chain PCR contained 6.5 ml of the
first-round reaction, 15 ml of PrimeSTAR Max Premix (23), 0.8 ml of each
forward primer, and 0.8 ml of each reverse primer. The first-round pro-
cedure was as follows: 98˚C for 2 min; 50 cycles of 98˚C for 10 s, 56˚C for
5 s, and 72˚C for 5 s; and finally, 72˚C for 5 min. The second-round
procedure was as follows: 98˚C for 2 min; 40 cycles of 98˚C for 10 s,
60˚C for 5 s, and 72˚C for 5 s; and finally, 72˚C for 5 min.
L chain k. The first round of L chain k PCR contained 6 ml of the RT
reaction, 15 ml of PrimeSTAR Max Premix (23), 1.0 ml of each forward
primer, and 1.0 ml of each reverse primer. The second round of L chain k
PCR contained 6 ml of the first-round reaction, 20 ml of PrimeSTAR Max
Premix (23), 1 ml of each forward primer, and 1 ml of each reverse primer.
The first-round PCR procedure was as follows: 98˚C for 2 min; 45 cycles
of 98˚C for 10 s, 54˚C for 5 s, and 72˚C for 5 s; and finally, 72˚C for 5 min.
The second-round procedure was as follows: 98˚C for 2 min; 45 cycles of
98˚C for 10 s, 60˚C for 5 s, and 72˚C for 5 s; and finally, 72˚C for 5 min.
Ab sequence evaluation, expression, and testing
The IGHV, IGKV, and IGLV subgroup distributions and the CDR3 length of
the IGV domain sequences were analyzed with IMGT/V-QUEST (http://
www.imgt.org). IgG expression vectors (pCMV-Rh) contain a monkey
Ig gene leader peptide and multiple cloning sites upstream of the monkey
Ig H chain, Ig kappa chain, and Ig lambda chain constant regions. The
methods for Ab expression in mammalian cells and purification with
Protein A are as follows. Briefly, a 3:2 ratio of the molar amounts of
H chain plasmid and L chain plasmid were cotransfected into 293T cells
for transient expression with Lipofectamine 2000 (Thermo Fisher Scien-
tific). The supernatants were harvested 4 d after transfection. The Abs were
purified with Protein A beads (Thermo Fisher Scientific) according to the
manufacturer’s instructions. The purified EV-D68 virion was analyzed by
SDS-PAGE. Briefly, the proteins were boiled for 10 min and then separated
on 10% Tris-glycine SDS-PAGE gels that were then stained with silver or
transferred to PVDF membranes for Western blot analysis. After blocking
with 5% nonfat milk in 0.1% Tween 20 in PBS (PBST) overnight at 4˚C,
the blots were incubated for 2 h at room temperature with A6-1 (4 mg/ml).
After washing in PBST, the PVDF membrane was detected by HRP-
conjugated rabbit anti-human IgG (1:5000; Abcam, Cambridge, MA) for
45 min at room temperature. The signals were visualized with ECL
Western blot substrate reagents.
ELISA
Briefly, the 96-well plates were coated with 0.2 mg/well recombinant VP1
from EV-D68 and incubated overnight at 4˚C. The plates were then
blocked with 5% dry milk in PBST at room temperature for 2 h. After three
washes with PBST, samples (polyclonal serum and expressed supernatant)
were added to the plates, which were then incubated for 2 h. After-
wards, the plates were washed five times with PBST and incubated for 1 h
with a rabbit anti-human IgG H&L HRP-conjugated secondary Ab
(Abcam). The assay was developed with TMB substrates (Solarbio Science
The Journal of Immunology
and Technology) according to the manufacturer’s instructions. The OD
were measured at 450 nm. Positive binding was defined as at least 2.5-fold
above the OD of the negative control. Binding capability was also tested with
the same ELISA with a 43 serial Abs dilution. For peptide mapping, the
plates were coated with 0.4 mg/well of each of the EV-D68 VP1 peptides at
4˚C overnight and blocked with PBST containing 5% BSA for 2 h at room
temperature, followed by 0.1 mg of A6-1 mAb (diluted in 100 ml of PBS) for
2 h at room temperature. After each time of incubation step, the plates were
washed five times with PBS. The OD were measured at 450 nm. Positive
binding was defined as at least 2.5-fold above the OD of the negative control.
Microneutralization assays
In this study, the neutralizing Ab titer produced by the two rhesus monkeys
was detected by a neutralization assay. Before this test, all serum samples
were inactivated at 56˚C for half an hour in a water bath. The cell culture
medium was formulated as follows: 10% new bovine serum, 1.5%
penicillin/streptomycin, 2% glutamine solution (3%), 3% NaHCO3 (6.6%),
and 83.5% MEM. We first placed 50 ml of the cell culture medium (2%
new bovine serum) in an empty 96-well plate and then added 50 ml of
2-fold diluted serum sample to each well of the first row. We diluted the
serum as follows: we transferred 50 ml of liquid from each well in the first
row to the corresponding well in the second row, then transferred 50 ml of
liquid from each well in the second row to the corresponding well in the
third row, and so on until the eighth row. We discarded 50 ml of liquid from
each well in the eighth row. Then, the diluted serum samples were incu-
bated for 1 h with 100 CCID50 of EV-D68 virus. HeLa cells digested with
2.5% trypsin were added to each well at a density of 2 3 104 cells/well and
cultured in a 37˚C incubator with 5% CO2. The cytopathic effect (CPE)
was tested for 7 d, and the CPE wells were counted daily.
Neutralization assays by 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
Neutralization assay. In the standard neutralization assay, 50 ml of A6-1
mAb was incubated with 50 ml of the EV-D68 KM strain (100 CCID50)
for 1 h at 37˚C; after which, the mixture was added to the HeLa cells.
The cells incubated with the mixture were cultivated for 72 h at 37˚C,
and the viability was detected with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent
(Beijing Solarbio Science and Technology). The neutralization efficiency
of each sample was calculated as follows: percentage of neutralization =
[(OD490 of each sample 2 OD490 of the cells treated with virus only)/
(OD490 of the cells untreated 2 OD490 of the cells treated with virus only)]
3 100. The IC50 represents the concentration of mAbs that inhibit cell
death by 50% (25).
Preattachment neutralization assay. The mAbs and virus were mixed and
incubated at 37˚C for 1 h, and the mixture was then incubated with the cells
for 2 h at room temperature. Next, the cells were washed three times with
PBS and then cultivated at 37˚C for 72 h. The cell viability and neutralization
efficiency for each sample was determined as previously described (25).
Postattachment neutralization assay. First, 50 ml of the EV-D68 KM strain
(100 CCID50) in DMEM was added to the HeLa cells at room temperature
for 1 h so that the virus could attach to the cell surface. The cells were
washed three times to remove unbound viruses and were then incubated
with 50 ml of A6-1 at 37˚C for 1 h. The cells were then washed three times
with PBS. After 72 h, the cell viability and the inhibitory efficiency were
determined as previously described (25).
Neutralization inhibition assay. The inhibition of the mAb-mediated
neutralization of EV-D68 infection by synthetic peptides (peptide 1 and
peptide 25) was determined by a neutralization inhibition assay. Briefly,
different amounts of peptides and A6-1 mAbs were mixed together for 1 h at
37˚C. The mixtures were incubated with 100 CCID50 of the EV-D68 KM
strain virus for 1 h at 37˚C. The A6-1-peptide/virus mixture was added in
the cells. After incubation at 37˚C for 72 h, the neutralization inhibition
assays were evaluated using an MTS-based kit (Beijing Solarbio Science
and Technology). The OD was determined at OD490 with a microtiter plate
reader. The neutralization inhibition efficiency (100%) = {1 2 [(OD490 of
each sample 2 OD490 of the cell treated with virus only)/(OD490 of the
cells untreated 2 OD490 of the cells treated with virus only)]} 3 100.
Cross-reaction neutralization assay. The experimental procedures were the
same as those of the microneutralization assays evaluated by CPE.
Laser confocal microscopy and quantitative PCR analysis of
the inhibition of virus attachment by A6-1
Different amounts of A6-1 were mixed with 106.5 CCID50 of EV-D68 virus
in DMEM (containing 2% FBS) and then incubated at 37˚C for 1 h before
2559
being added to Vero or HeLa cells (2 3 105 cells/well in 12-well plates).
The cells were incubated with the A6-1/virus mixture for 2 h at room
temperature. After we wash the cells three times, the cell-attached viruses
were used for qRT-PCR and immunofluorescence analysis. For quantifi-
cation of viral loads by qRT-PCR, the cells attached to the EV-D68 virus
were harvested for extracting viral RNA extraction; EV-D68 viral RNA copies
were detected using the same procedure described in the section on viral load
detection in rhesus monkey infection. For immunofluorescence analysis, slides
of the cells were incubated with 4 mg/ml A6-1 labeled with the Lightning-Link
Fluorescein Conjugation Kit (Innova Biosciences) according to the protocols
provided in the kit and 5 mg/ml biotinylated Sambucus nigra lectin (a2,6-
linkage) (Vector Laboratories) at room temperature for 2 h. Then, the slides
were washed three times with PBS and incubated with 5 mg/ml rhodamine
avidin (Vector Laboratories) for 1 h at room temperature. Finally, the slides
were observed by confocal laser microscopy.
Fluorescence resonance energy transfer/fluorescence
lifetime imaging and stimulated emission depletion
microscopy imaging
PCR-amplified EV-D68 VP1 gene fragments (bp 1–453, bp 301–750, and
bp 628–927) were cloned into the pSNAPf Vector (New England Biolabs).
The A6-1 mAb plasmid was cotransfected into 293T cells with the above
VP1 fragment plasmids. The fluorescence lifetime imaging (FLIM)/
Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021
fluorescence resonance energy transfer (FRET) and stimulated emission
depletion (STED) microscopy imaging experiments were performed 36 h
after transfection. After 36 h of transfection, a labeling medium consisting
of 3 mM SNAP-Cell TMR-Star dye (New England Biolabs) was used to
replace the medium on cells expressing a SNAP fusion protein, which were
then incubated at 37˚C with 5% CO2 for 10 min. The cells were then
washed three times with complete culture medium containing 10% FBS
and were then incubated in fresh medium for 30 min. Next, the medium
was replaced once to remove unreacted SNAP-tag substrate that had dif-
fused out of the cells. Then, the cells were fixed with 4% paraformalde-
hyde (Beijing Solarbio Science and Technology) for 15 min at room
temperature and rinsed three times with PBS. Next, the cells were per-
meabilized with 0.1% Triton in PBS for 10 min, washed three times with
PBS, and then blocked with 10% FBS in PBS overnight at 4˚C. The next
day, the cells were incubated with goat anti-human IgG Fc (DyLight 488)
(Abcam) for 1 h at room temperature. Finally, the cell nuclei were coun-
terstained with DAPI (Abcam), and the slides were kept at 4˚C until use.
For FLIM/FRET experiments (26), the “donor only” sample consisted of
the same cells transfected with only the A6-1 mAb plasmids. Then, the
average fluorescence lifetimes of the donor-only and FRET samples were
measured in fast FLIM mode. The measurement was completely controlled
via Leica Application Suite Advanced Fluorescence. The FRET efficiency
was calculated from the ratio of the FRET donor lifetime tquench and the
non-FRET lifetime tquench as follows:
F RET efficiencyð%Þ ¼ 1 2 ðtdonor ; F RET Þ=ðtdonor ; no F RET Þ
For the STED microscopy imaging, the cell slides were visualized and
captured with an appropriate filter set under a Leica TCS SP8 microscope
equipped with a STED laser (Leica Microsystems). Finally, we analyzed
the colocation between the A6-1 mAb and different VP-truncated proteins.
Mouse protection experiments
The mice protection experiments were approved by the IACUC of the IMB,
CAMS. VRC01 used as the irrelevant Ab in this study is a neutralizing
human mAb against HIV-1 (27). Three groups of 2-d-old suckling mice
were i.p. injected, respectively, with purified A6-1 Ab (30 mg/mouse),
VRC01 (30 mg/mouse) as irrelevant Ab, or PBS 12 h before the nasal
administration of the virus. For viral infection, 10 ml of the EV-D68 KM
strain (5 3 105 CCID50/ml) was micropipetted onto the noses of each
suckling mouse two times with a 30-min interval between exposures.
Then, three mice in each group were randomly sacrificed at four time
points (0, 3, 7, and 10 dpi) to measure the viral load. The lung and brain
tissues of the mice were sampled for histopathological examination and
immunofluorescence analysis at 0, 3, and 7 dpi.
Viral load determined by virus titration assay
EV-D68 acquired from lung or brain tissues of virus-infected suckling mice
were analyzed by a microtitration assay (28). In brief, the virus supernatant
was extracted from lung and brain homogenates. Then, the supernatants
were passed through a 0.22-mm sterile filter, and HeLa cells in 96-well
plates were inoculated and cultivated with the supernatants at 37˚C for 7 d.
The CPE was observed each day.
2560 A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION

Histopathological examination and immunofluorescence
analysis of mouse brain and lung tissues
The brain and lung tissue samples were embedded in an optimal cut-
ting temperature compound and frozen in liquid nitrogen. The frozen
tissues were cut into 6-mm sections and were placed on the poly-i-lysine–
coated glass slides and fixed in 4% paraformaldehyde. The glass slides
containing tissue samples were stained with H&E according to the manu-
facturer’s instructions (Beijing Solarbio Science and Technology). For
the EV-D68 viral immunofluorescence analysis, the prepared frozen sections
were stained with anti–EV-D68 VP1 Ab (GeneTex), followed by a goat anti-
rabbit secondary Ab labeled with Alexa Fluor 647 dye (Thermo Fisher
Scientific) and observed by confocal microscopy (Leica Microsystems) (29).
Results
Characterization of EV-D68 infection in rhesus macaques
We established an animal model of Chinese rhesus macaques infected
with the KM strain of EV-D68. Two 6-mo-old rhesus macaques were
successfully infected via the nasal tract (104.5 CCID50/monkey). To
assess the Ab responses against EV-D68 in these two animals, plasma
samples harvested during a viral exposure challenge were assayed for
their neutralizing activity. The viral replication was typical of EV-D68
replication during a primary infection (23, 30), and the virus replication
kinetics did not differ significantly between these two animals.
We observed that the viral loads in both rhesus monkeys began to
increase on the third day after EV-D68 infection. The average viral
load in the blood peaked at 4740 copies/ml 9 dpi (Fig. 1A).
However, the viral loads in the nasal washes and feces peaked on
the fifth day, at 14,866 copies/100 mg of feces and 2697 copies/ml
in the nasal washes, and then decreased rapidly after the seventh
day (Fig. 1B). Either the viremia in the blood or the high viral load
in the nasal swabs and feces was sufficient to indicate that both
monkeys were infected by EV-D68, although the clinical features
were slight (data not shown).
For the immune reaction induced by the viral infection, the neu-
tralizing Ab against EV-D68 was detected positively 14 dpi in both
rhesus monkeys, with a titer of 23 and 24 (Fig. 1C), whereas the total
Ab binding to EV-D68 as analyzed by ELISA also showed increasing

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

FIGURE 1. The establishment of an EV-D68–infected rhesus monkey model and single memory B cell sorting. Dynamic profiles of EV-D68 in (A) the blood
and (B) the nasal swabs and feces of EV-D68–infected rhesus monkeys. EV-D68 viral RNA extracted from these samples was detected using TaqMan real-time
quantitative PCR. The dotted line represents the SEM of viral load changes in feces; the solid line represents the SEM of viral load changes in nasal swab. (C)
Neutralizing Ab responses to EV-D68 virus after 0, 7, 14, and 28 dpi. The neutralization Ab titer was the reciprocal of the highest serum dilution that inhibited
50% of the viral CPE. (D) Reactivity of the anti–EV-D68 sera from the infected monkeys against the EV-D68 VP1 protein after 0, 7, 14, and 28 dpi; the ab-
sorbance value was measured at 450 nm. The results presented are representative of three independent experiments. (E and F) Single-cell sorting of EV-D68 VP1
memory B cells by flow cytometry. The numbers in the density plots indicate the percentages of cells gated from the previous gate side scatter (SSC).
The Journal of Immunology 2561

Table I. The first and second nest PCR primer list

Primer Name Primer Sequence

First round H chain primer 59 primer VH1A 59-TCSTCTCCACAGGCGCCCACTC-39

VH1B 59-TCCTCTMCRYAGGTGCCMASTC-39
VH1C 59-TCCTCTCCGCAGGGGCCCACTC-39
VH2 59-GTCCCGTCCTGGGTCTTGTC-39
VH3A 59-CTATTTTARRAGGTGTCCAGTG-39
VH3B 59-CTCTTTTGAAAGGTGTCCAGTG-39
VH4 59-AGCTCCCAGATGGGTCYTGTCC-39
VH5 59-TCTCCCCCACAGGAGTCTGTGC-39
39 primer Reverse 1H 59-GGACAGCCKGGAAGGTGTGC-39
First round l chain primer 59 primer VL 1 59-GGTCCTGGGCCCAGTCTGTGCTG-39
VL 2 59-GGTCCTGGGCCCAGTCTGCCCTG-39
VL 3 59-GCTCTGTGACCTCCTATGAGCTG-39
VL 4 59-GGTCTCTCTCSCAGCYTGTGCTG-39
VL 5 59-GTTCTTGGGCCAATTTTATGCTG-39
39 primer Reverse 1L 59-CACCAGTGTGGCCTTGTTGGCTTG-39
First round k-chain primer 59 primer Vk1 59-TCCAATYTCAGGTGCCARATGT-39
Vk2 59-ATTTCAGGATCCAGTGGGGAT-39
Vk3 59-TCCAATTTCAGATACCACYGGA-39
Vk4 59-TGGGTCTCGGTGCCCGTCAGG-39
59-TGGATCTCTGGTGCCTGTGGG-39

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

Vk5
Vk6 59-TGTGCTCCAGGCTGCAATGGG-39
39 primer Reverse 1K 59-GAGGCAGTTCCAGATTTCAA-39
Second round H chain primer 59 primer VH1A 59-GAGGTCCAGCTGGTRCAGTC-39
VH1B 59-CAGGWGCAGCTGGTGCAGTC-39
VH2 59-CAGGTGACCTTGAAGGAGTCTG-39
VH3 59-GARGTGCAGYTGGTGGAGTCTG-39
VH4A 59-CAGSTGCAGCTGCAGGAGTCGG-39
VH4B 59-CAGCTGCAGCTGCAGCTGCAGG-39
VH5 59-GAGGTGCAGCTGGTGCAGTCTG-39
VH6 59-CAGGTGCAGCTGCAGGAGTCAG-39
VH1 59-GAGGAGACGGTGACCAGGGC-39
VH2 59-GAGGAGATGGTGATTGGGGT-39
VH3 59-GAAGAGACGGTGACCCTGAG-39
VH4 59-GAGGAGACGGTGACCAGAAC-39
VH5 59-GAGGAGACGGTGACGACGAC-39
39 primer Reverse-VH1 59-GAGGAGACGGTGACCAGGGC-39
Reverse-VH2 59-GAGGAGATGGTGATTGGGGT-39
Reverse-VH3 59-GAAGAGACGGTGACCCTGAG-39
Reverse-VH4 59-GAGGAGACGGTGACCAGGAC-39
Reverse-VH5 59-GAGGAGACGGTGACCAGAAC-39
Reverse-VH6 59-GAGGAGACGGTGACGACGAC-39
Second round l chain primer 59 primer VL 1 59-TCCTGGGCCCAGTCTGTGCTGACKCAG-39
VL 2 59-TCCTGGGCCCAGTCTGCCCTGACTCAG-39
VL 3 59-TCTGTGACCTCCTATGAGCTGACWCAG-39
VL 4 59-TCTCTCTCSCAGCYTGTGCTGACTCA-39
VL 5 59-TCTTGGGCCAATTTTATGCTGACTCAG-39
39 primer Reverse 2L 59-CTCCTCAGCTAGCGGYGGGAACAGAGTG-39
Second round k-chain primer 59 primer Vk1 59-GACATYCAGATGWCCCAGTCTC-39
Vk2A 59-GATAYTGTGATGAYCCAGACTC-39
Vk2B 59-GATGTTGYRATGACTCAGTCTC-39
Vk3A 59-GAAATWGTRATGACGCAGTCTC-39
Vk3B 59-CAAGTTATATTGACTCAGTCTC-39
Vk4 59-CTGGATCTCTGGTGTCTGTGG-39
Vk5 59-CCTTTGGATCTCTGMTGCCAGG-39
39 primer Reverse-Vk1 59-TTTGATCTCCAGCTT-39
Reverse-Vk2 59-TTTGATTTCCACCTT-39
Reverse-Vk3 59-TTTGATCTCCACTTT-39
Reverse-Vk4 59-TTTGATATCCAGTTT-39
Reverse-Vk5 59-TTTAATCTCCAGTCG-39
K = G + T; M = A + C; R = A + G; S = G + C; W = A + T; Y = C + T.

OD450 values of 0.375 and 0.225 (Fig. 1D). These results are similar
to our previous findings in EV-D68–infected ferrets (23).
Identifying VP1-specific memory B cells against EV-D68 and
single B cell sorting
Based on the neutralizing Ab responses, we performed FACS on
PBMCs from donor monkeys 15247 and 15249. Compared with the
very small percentage of memory B cells (CD20+/CD27+/EV-D68
VP1+) detected in monkeys before EV-D68 infection, ∼3.56 and
2.02% of the memory B cells from donor monkeys 15247
(Fig. 1E) and 15249 (Fig. 1F), respectively, were VP1 specific,
indicating an obvious increase in EV-D68 Ag presentation by
BCR after EV-D68 infection.
The ability of sorted Abs to bind EV-D68
To determine the Ab properties of the isolated memory B cells, we
amplified the Ab gene by cDNA synthesis from single memory
B cells followed by nested PCR (24, 31, 32). Two rounds of
specific PCR were performed with two sets of optimal primers
for monkey IgG genes (Table I). Of the 301 single cells tested,
2562 A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION

31 cells produced both H and L chain products, representing a
10.3% cloning efficiency among the positive wells.
To assess the proportions of cloned IgGs that could recognize
EV-D68 immunogens, we expressed these 31 IgGs in a 293T cell
system. All mAbs secreted into the cell culture supernatant were
used for the VP1-binding ELISA screening (Fig. 2A). Of the total
Abs tested, A6-1, E2-2, F4-3, and D7-4 showed obvious positive
Ag binding against purified EV-D68 VP1. We then assessed
whether A6-1 could bind to KM EV-D68 viral proteins by Western
blot analysis because A6-1 bound to VP1 with the highest bind-
ing capability. The results demonstrated that mAb could bind to
EV-D68’s VP1 with specificity, indicating that the A6-1 candi-
dates could also recognize viral particles (Fig. 2B).
Neutralizing activity against EV-D68
We next explored the EV-D68–neutralizing capacity of these four
Ab candidates. A6-1 fully neutralized two genotypes of the EV-D68
strain (Fermon as genotype A and KM as genotype B), achieving
100% inhibition, with IC50 values of 0.6 mg/ml for the KM strain
with 36–37% of the mutations in the VH. F4-3 and A6-1 possess
18–19% of the L VL, which is different from the speculative
germline gene sequences; the L chain of E2-2 was derived from a
different germline, VK4.1. All three Abs contained a relatively
long CDRH3 loop of 21–22 aa (Fig. 3C) and mutations at residue
F97 and S109 in A6-1 (Fig. 3C), which likely introduced potential
differences into their binding affinities. The results of ELISA in-
dicated that A6-1 still displayed efficient affinity to VP1 with a
low concentration of 0.05 mg/ml compared with the other three
mAbs (Fig. 3D). We also noticed that A6-1 and F4-3 shared
the same germline with only 2 aa differences in the H chain.
Moreover, compared with F4-3, the homology-modeling three-
dimensional structure of A6-1 indicated an S109 deletion in
CDRH3, forming a longer loop that affected its neutralizing
activity (Fig. 3E).
The H chain of D7-4 was derived from a different progenitor,
VH1.36, but its L chain was derived from the same VL3.10 alleles
as A6-1. D7-4 had a lower somatic mutation rate, with 15% of
VH and 19% of VL nucleotides different from the putative germ-

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

(Fig. 3A) and 1.57 mg/ml for the Fermon strain (Fig. 3B). A6-1
exhibited greater potency than the F4-3, E2-2 and D7-4 mAbs,
which all had similar IC50 values against both strains (in the range
of 2–6 mg/ml); none of these three Abs achieved a maximum
percent inhibition (MPI) of 100%.
Notably, the H chain complementarity-determining regions
(CDRHs) of mAbs F4-3, A6-1, and E2-2 pertain to the progeni-
tor IGHV3.9. An analysis of the variable Ab genes revealed that
these three pairs of Abs shared almost identical somatic mutations,
line gene sequences. The CDRH3 of D7-4 contained 14 aa.
Comparatively, A6-1 from the progenitor IGHV3.9 showed the
best neutralizing activity against EV-D68, suggesting its high-
lighted potency for our further study (Fig. 3C).
Long CDRH3 loops are relevant to Ab specificity and cross-
reactivity (33); therefore, we tested all four Abs for reactivity
against a panel of two genotypes of EV-D68 and two other
enteroviruses, EV71 and CA16. None of these four Abs showed
any substantial binding capability or neutralization activity toward

FIGURE 2. Affinity and specificity of the EV-D68 VP1–sorted paired mAbs. (A) The affinities of the isolated EV-D68–specific mAbs for EV-D68 VP1
were determined by screening. Purified EV-D68 KM strain VP1 protein was coated onto 96-well plates overnight at 4˚C. The absorbance at 450 nm was
measured. The Abs were substituted for PBS in the control group. The positive sample values were defined by OD450 . 0.1 and OD450 . 2.5-fold of OD450
value of negative control. The dotted line shows the OD450 is 0.1. The results presented represent data from three independent experiments (B). Silver stain
analysis of the purified Ags. The first lane is the marker, and the second lane shows the main structural proteins of the EV-D68 viral particles, including VP0
and VP1. Western blot analysis: purified EV-D68 virus was resolved on a 10% SDS-PAGE gel, transferred to a PVDF membrane and probed with A6-1
mAbs, followed by incubation with rabbit anti-human IgG (HRP) secondary Abs.
The Journal of Immunology 2563

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

FIGURE 3. Characterization of F4-3, A6-1, E2-2, and D7-4. (A and B) The Neutralization activity of the mAbs against EV-D68: (A) EV-D68 KM strain
and (B) EV-D68 Fermon strain. The values in parentheses represent the mAb IC50 (micrograms per milliliter). The IC50 was calculated using GraphPad
Prism 5. The dotted line shows 50% of neutralization. The data were acquired from three independent experiments. (C) The amino acid sequences of the
variable regions of F4-3, A6-1, E2-2, and D7-4 mAb. Partial framework (FR) and CDRHs are provided above. The first sequence of each group represents
the germline sequence. F4-3, A6-1, and E2-2 were derived from the same VH germline gene (VH3.9), whereas D7-4 was derived from VH1.36, F4-3, A6-1,
and D7-4 and had the same VL gene (VL3.10), whereas the gene sequence of E2-2 was matched with the germline sequence (VK4.1). The amino acid
residues labeled in red indicate the mutation compared with the germline sequence. The amino acid residues labeled in blue indicate the germline sequence.
(D) Binding activities of the mAbs. EV-D68 VP1 protein were coated onto the 96-well plates and were examined for their reactivity to the different
concentrations of A6-1, E2-2, D7-4, and F4-3 by ELISA. The data were acquired from three independent experiments. ns, p $ 0.05; *p value , 0.05 was
considered significant. (E) A three-dimensional diagram of F4-3 and A6-1. CDRH3 regions are shown in yellow. The blue contained in the yellow rep-
resents the loop region in CDRH3. The purple represents the residues Leu of F4-3 and Phe of A6-1 in the 97. The F4-3 and A6-1 three-dimensional
structure models in this panel were predicted using the online software SWISS-MODEL. (F) Cross-binding and activity of F4-3, A6-1, E2-2, and D7-4
mAbs in the CA16 and EV71 strains. The EV-D68 KM strain VP1 protein and two purified inactivated viruses (CA16 and EV71) were examined for their
reactivity with the mAbs by ELISA. The positive sample values were defined by OD450 . 0.1 and OD450 . 2.5-fold of OD450 value of negative control.
The dotted line shows that the OD450 value is 0.1. The data were acquired from three independent experiments.

the EV71 or CA16 strains tested, confirming the specificity of donor fluorophore when an acceptor fluorophore exists because of
these Abs to target EV-D68 (Fig. 3F, Table II). energy transfer. Therefore, by immunostaining a VP1-truncated
protein (acceptor) and an A6-1 mAb (donor) with fluorophores
A6-1 displayed a better affinity toward the EV-D68 VP1 region at emission wavelengths of 580 and 524 nm, respectively, the
of 101–151 aa distance between the VP1-truncated protein and the A6-1 protein
We speculated that A6-1, E2-2, and F4-3 might recognize the same can be detected by FLIM through calculating the fluorescence
or similar epitopes because these three Abs are somatic variants
with many similarities. As a representative, we used A6-1 to ex-
amine the binding affinity with VP1 subregions (VP1-1, aa 1–151; Table II. EV-D68 mAb cross-reactivity by neutralization assay
VP1-2, aa 101–250; and VP1-3, aa 210–309) by STED. Confocal
imaging showed that A6-1 bound strongly to EV-D68 VP1-1 and Inhibition of CPE by mAb Treatment
VP1-2, which both overlap with the DE loop region at 101–151 Enteroviruses E2-2 A6-1 F4-3 D7-4
aa, but did not bind strongly to VP1-3 (Fig. 4A, 4B).
EV-D68 (Fermon) + + + +
We next investigated if the great neutralization potency of A6-1 EV-D68 (KM) + + + +
resulted in a high binding affinity to specific Ags VP1, VP1-1, EV-71 2 2 2 2
VP1-2, and VP1-3 of the EV-D68 Fermon and KM strains using CA-16 2 2 2 2
a FLIM/FRET assay (27, 34–36). FRET can be observed in the Samples with 100% cells free of CPE were considered positive (+), otherwise
FLIM/FRET process; FRET activity shortens the lifetime of the negative (2).
2564 A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

FIGURE 4. A6-1 recognized VP1-1 (aa 1–151), located at the viral DE loop region. (A and B) The A6-1 mAb plasmid was cotransfected into 293T cells
with VP1 fragment plasmids (VP1-1, aa 1–151; VP1-2, aa 101–250; and VP1-3, aa 210–309). Red represents the expressed EV-D68–truncated VP1
proteins VP1-1, VP1-2, or VP1-3. Green represents A6-1 interacting with the different VP1 proteins. Yellow represents the merged image. Images are
shown at original magnification 3100. (C and D) FLIM/FRET technology illustrates the interaction between EV-D68 VP protein (acceptor) and A6-1
(donor). Negative FRET capture control: donor only (only A6-1) without acceptor, VP1 + A6-1 (EV-D68 Fermon full-length VP1 + A6-1), VP1-1 + A6-1
(EV-D68 Fermon VP1-1 + A6-1), VP1-2 + A6-1 (EV-D68 Fermon VP1-2 + A6-1), and VP1-3 + A6-1 (EV-D68 Fermon VP1-3 + A6-1). The FRET
efficiency (%) was calculated using the formula provided in Materials and Methods. Each histogram value represents (Figure legend continues)
The Journal of Immunology 2565

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

FIGURE 5. Understanding the neutralization of A6-1. (A) Inhibition of A6-1–mediated neutralization by peptides 1 and 25. The neutralizing inhibition of the peptides
was determined by an MTS assay. The neutralization inhibitory efficiency (100%) is represented as the mean 6 SEM of three independent experiments. (B and C) The cell-
attached EV-D68 virus was analyzed by qRT-PCR. The dotted line indicates the highest OD450 value in these assays. The data are represented as the mean 6 SEM of three
independent experiments. ns, p $ 0.05; *p value , 0.05 was considered significant. (D and E) The cell-attached EV-D68 virus was analyzed by confocal microscopy. The
control (top) represents the cells without treatment. The experimental group (middle) represents cells incubated with the EV-D68 KM strain virus only. The experimental
group (bottom) represents the cells incubated with an A6-1/virus mixture. The EV-D68 virus and a2,6-linked sialic of cells in all groups were colocated by confocal
microscopy. EV-D68 was detected by A6-1–labeled FITC (green), and the a2,6-linked sialic acids on the cell surface were incubated with biotinylated Sambucus nigra lectin
(a2,6-linkage), followed by the secondary Ab with rhodamine dye (red). The overlaid image (orange) of the two makers reveals a strong colocation of the a2,6-linked sialic
acid on EV-D68–positive cells. Images are shown at original magnification 363. (F and G) A6-1 inhibited EV-D68 infection at the preattachment and the postattachment
stage. The neutralization efficacies of A6-1 at different concentrations during the pre- or postattachment stages. The data are represented as the mean 6 SEM of three
independent experiments. (H) EV-D68 was preattached to HeLa cells and then treated with A6-1. A6-1 was then detected by rabbit anti-human IgG Fc labeled with APC
dye and observed by confocal microscopy. The red color shows the EV-D68 virus attached to the cells. Images are shown at original magnification 363.

lifetimes of the donor fluorophores. We can measure the proximity efficiency values of A6-1 for KM VP1, VP1-1, VP1-2, and VP1-3
between two proteins by calculating the FRET efficiency, reveal- were 30.3, 33.7, 14.8, and 5%, respectively (Fig. 4C), and the
ing the characteristics of protein/protein interaction. The FRET FRET efficiency values of A6-1 for Fermon strains VP1, VP1-1,

the average FRET efficiency (%) (6SD) from five individual cells. The images at the bottom of the histogram represent the intensity images from the donor
channel and the lifetime map for one representative cell expressing different-length VP1 proteins. Images are shown at original magnification 363. (E) EV-D68
VP1 peptide mapping by ELISA. Sixty peptides, spanning the EV-D68 VP1 region, were used to screen A6-1 by ELISA. The positive value is defined as OD450
. 0.1. The data are presented as the mean 6 SEM of the OD450 readings from three different independent experiments. (F) The 15 peptides of the Fermon VP1
protein that differ from the KM VP1 protein were also screened for reactivity to the A6-1 by ELISA. The dotted line indicates the highest OD450 value in these
assays. The data are presented as the mean 6 SEM of the OD450 readings from three different independent experiments. (G) Identification of the similar amino
acid sequences among peptides 24, 25, and 26 and the DE loop of EV-D68 VP1. The similar sequence bound by A6-1 is highlighted in red. (H) Binding of A6-1
to ID 25 peptide mutants. A panel of nine peptides was subjected to residue-by-residue mutagenesis to Ala from aa 126 to aa 135. Wild type (WT) represents
the original ID 25 peptide without any mutation. The data were acquired from mean 6 SEM of the OD450 readings from three independent experiments.
2566
FIGURE 6. Three-dimensional diagram of the interaction between the EV-D68 main structure VP1 (yellow), VP2 (light blue), and VP3 (purple) protein
complexes and the A6-1 mAb. The A6-1 mAb (green) binds to the DE loop (red) of EV-D68 VP1. The amino acid residues labeled in blue in the dashed
circle, including Asn275 and Arg270 of VP1 and Arg104, Asp232, Pro231, Asp91, Arg95, and Ile233 of VP3, make up the binding site of the sialic acid receptor.
The crystal structure of EV-D68 was obtained from the Protein Data Bank (PDB; accession number 4WM8, http://www.rcsb.org/). The A6-1 three-di-
mensional structure models in this article were predicted using the online software SWISS-MODEL.
VP1-2, and VP1-3 were 21.5, 21.2, 14, and 2.4%, respectively
(Fig. 4D). At the same time, the change in the pseudo-color im-
ages of A6-1 (donor) from green to blue demonstrated that the
donor’s fluorescence lifetime decreased with increasing FRET
efficiency (Fig. 4C bottom, Fig. 4D bottom). The FRET efficiency
values showed that the distances between the A6-1 mAb and VP1
or VP1-1 from different strains were less than those of the other
truncated VP1 proteins, demonstrating that A6-1 had a better af-
finity for VP1-1 and possibly explaining why it had the highest
neutralizing activity against EV-D68.
A6-1 recognized EV-D68 VP1-1 (aa 1–151) in epitope
mapping and located the viral DE loop region
To further map the A6-1 epitope within VP1, we screened a panel of
60 overlapping peptides spanning the sequences of the entire VP1
gene of the EV-D68 KM strain for reactivity. As shown in Fig. 4E,
A6-1 reacted strongly with peptide 25 (aa 121–135) but less so
with other peptides. Considering certain differences in the se-
quences of the BC, DE, and GH loops in the EV-D68 Fermon
strain VP1 gene, we also screened an extra panel of 15 peptides
covering these regions of the Fermon strain (Fig. 4F). The A6-1
also bound to the same region with the sequence RFDAEITILT
(Fig. 4G), which harbors the neutralizing immunogen sites
NIm-IA and NIm-IB (22).
To assess the importance of individual binding site residues for
the epitope recognition of the A6-1 Ab, alanine scanning analysis
was performed against peptide 25. As shown in Fig. 4H, alanine
substitution at residues 127, 128, 130, 132, 134, and 135 did not
strongly affect the binding of the corresponding peptides to
A6-1, whereas point mutations at positions 126, 131, and 133
weakened A6-1 binding considerably, indicating that the three
amino acids R126, I131, and I133 were crucial for A6-1 binding
(Fig. 4H). Considering that A6-1 recognized the VP1-1 in cells
and bound the DE loop in the epitope screening, we speculated
that the specific peptide corresponding to the DE loop affected
the A6-1–mediated neutralization of the KM and Fermon strains
of EV-D68.
Understanding the neutralization against EV-D68 of A6-1
To determine whether these Ab-binding peptides were indeed
involved in Ab recognition and function, two representative pep-
tides, 1 and 25, were used to block A6-1 neutralization. As shown in
Fig. 5A, a low concentration (2.5 mg/ml) of peptide 25 signifi-
cantly reduced the neutralization activity of A6-1, whereas as a
control sample, peptide 1 had no significant inhibitory effect, even
at the highest concentration used (20 mg/ml) (Fig. 5A).
A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION
To further understand the potential mechanism of the inhibitory
Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021
functions of A6-1, we investigated whether A6-1 could block the
binding of EV-D68 to its receptors. An a2,6-linked sialic acid is an
attachment receptor for EV-D68 (22, 37), and we examined whether
A6-1 treatment could interfere with the interaction between the
virus and the receptor. A6-1 treatment potently reduced the amount
of the virus pulled down in an Ab dose-dependent pattern, resulting
in an up to 80% decrease when ∼2.5 mg of A6-1 was incubated with
HeLa cells (Fig. 5B). A similar trend of Ab dose-dependent inhi-
bition was also observed in Vero cells (Fig. 5C), indicating that
A6-1 can interfere with EV-D68 binding to its a2,6-linked sialic
acid receptor and thereby inhibit virus entry. In the different cell
types (HeLa or Vero cells) treated with the virus only (Fig. 4D,
4E middle), the cell surface was surrounded by the EV-D68 virus
(green), whereas the amount of virus attached to the cells signifi-
cantly decreased when the cells were treated with the A6-1 and
virus mixture (Fig. 4D, 4E bottom). This indicated that A6-1 pre-
vented the adhesion of the EV-D68 virus to Vero and Hela cells.
Moreover, colocalization of the virus to the a2,6-linked sialic acid
(red) showed that A6-1 reduced the attachment of the virus to the
a2,6-linked sialic acid. In conclusion, A6-1 interferes with the at-
tack of EV-D68 on a2,6-linked sialic acid (Fig. 4D, 4E).
To more comprehensively understand the possible multiple
molecular mechanisms by which A6-1 inhibits EV-D68, we fur-
ther examined the preattachment and postattachment effects by
preincubating the Ab and virus together before applying them to the
cells and by applying the Ab to the cells pi with the virus. The Ab
pretreatment clearly rendered the virus highly sensitive to A6-1 in
the KM strain, with an IC50 of 1.39 mg/ml (Fig. 5F), whereas
posttreatment did not exhibit any neutralization (Fig. 5G) even
though A6-1 bound to the virion attached to the target cells
(Figs. 5H, 6). This indicated that the Ab did not inhibit the virus
with postattachment neutralization manner. Together, these data
show that the primary anti–EV-D68 activity of A6-1 arises from
blocking the binding of the virus to its receptor.
Animal protection
EV-D68 causes infection and death in some reports of mouse in-
fection models (38–41). Thus, we evaluated the protective efficacy of
A6-1 in 2-d-old suckling C57BL/6 mice. After nasal challenging with
EV-D68 KM strain, the virus was undetectable from 0 through 10 dpi
in the mice treated with A6-1. However, in the PBS or irrelevant Ab–
treated group, the virus can be detected in the lung and brain tissues
at 3 dpi and showed a further increasing at 7 dpi and then declined,
becoming undetectable at 10 dpi (Fig. 7A, 7B). Meanwhile, com-
pared with the A6-1–treated mice, in histopathological changes such
The Journal of Immunology 2567

Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021

FIGURE 7. A6-1 protects suckling mice from EV-D68 intranasal infection. Two-day-old suckling C57BL/6 mice were treated with PBS, A6-1 (30 mg/
mouse), or VRC01 Abs (serve as the irrelevant Ab control, 30 mg/mouse) 12 h before intranasal viral administration with purified KM strain EV-D68 and were
then monitored daily for 10 d. The mice were injected with nothing in the blank group. Three mice from each group were randomly sacrificed at four time
points (0, 3, 7, and 10 dpi) for viral load detecting. Immunofluorescence analysis and H&E staining was done at 0, 3, and 7 dpi. (A) The viral load in lung and
brain tissue was detected by viral titer assay (n = 15 per group). The viral titers are represented as the mean 6 SEM of three independent experiments. (B)
Immunofluorescence analysis of EV-D68 virus distributed in the lung and brain tissues from mice treated with A6-1, PBS, or VRC01 at 3 and 7 dpi. The viral
Ag was detected by anti–EV-D68 VP1 Ab, followed by secondary Ab labeled with Alexa Fluor 647 dye and observed under confocal microscopy. EV-D68
virus was displayed in red. Images are shown at original magnification 363. (C) Analysis of histopathological changes in the lung and brain tissues from mice
treated with A6-1, PBS, or VRC01 at 3 and 7 dpi. Infiltration of inflammatory cells (blue arrow), hemorrhage (black arrow), degeneration of lung tissue (red
arrow), and accumulation of the glial cells (orange arrow) were indicated in images. Images are shown at original magnification 320.

as hemorrhaging, inflammatory cells aggregation were observed in
the lung tissue at 3 or 7 dpi in PBS or irrelevant Ab–treated group.
Similar results were found in brain tissue, with an observation of
infiltration of inflammatory cells and accumulation of the glial cells at
3 or 7 dpi (Fig. 7C). No obvious pathological changes were observed
in the brain tissue of the mice treated with A6-1 (Fig. 7C). These
results indicated that A6-1 inhibited the EV-D68 intranasal infection
and alleviated the pathological outcomes of the infected mice.
Discussion
We isolated a class of EV-D68 entry–blocking neutralizing Abs from
infected monkeys. Low concentrations of these mAbs neutralized
KM and Fermon strains in vitro. One mAb, A6-1, was also effective
in an EV-D68 intranasally infected mouse model. To the best of our
knowledge, no previous study has reported any mAbs that could
neutralize EV-D68 isolates.
In this study, we infected Chinese rhesus macaques with the EV-
D68 virus and isolated their mAbs via single memory B cell sorting
using FACS, RT-PCR, and nested PCR of their PBMCs after 14 d of
infection. Of the total isolated Abs induced by EV-D68 infection,
10.3% percent were specific to VP1.
The mAbs isolated in our study exhibited unique features and
different activities. The H chains of F4-3, A6-1, and E2-2 possess
extensive somatic mutation compared with the D7-4 Ab, resulting
in a higher affinity for VP1 of EV-D68. Moreover, A6-1 exhibited a
highly potent neutralizing activity, reaching 100% MPI. Previous
2568
studies showed that potent neutralizing activity is usually associ-
ated with a high affinity (42–44). The high affinities of the A6-1
identified in this study also render it effective against EV-D68.
We attribute the A6-1’s antiviral activity to its attack on the
EV-D68 receptor. The Ab binds with a high capacity to EV-D68
VP1-1, thereby interfering with its interaction with the target cell
surface for viral entry. Our results (Fig. 5D, 5E) and recent studies
(21, 23, 37, 45) show that EV-D68 prefers to recognize a2,6-
linked sialic acid receptors. According to recent research (21), the
structure of EV-D68 shows that its surface depression (canyon)
consists of VP1 protein, which is the site of receptor binding (22,
46, 47). Although the floor of the canyon formed by the GH loop
of VP1 and partial segments of VP3 is an important position for
viral attachment to the cell receptors (21, 48, 49), the BC loop and
DE loop of VP1 are also important to its binding, because both are
near the canyon and close to 5-fold axes (22, 50). In this study, the
A6-1–binding epitope AEITILT (aa 129–136), located in the DE
loop region (22), is conserved in A, B, and C clades of EV-D68
(22, 51). Whereas this region is near the binding site of the sialic
acid receptor that exhibited highly potent neutralizing activity
reaching 100% MPI, we conclude that A6-1 may interfere with
EV-D68, attaching to the a2,6-linked sialic acid host entry re-
ceptor by binding to the DE loop, causing a steric hindrance and
thereby achieving viral neutralization (Fig. 6).
The identification of the DE loop of EV-D68 as a primary epitope
suggests the possibility that the mechanism of action of A6-1
involves targeting a specific Ag location and causing a steric
clash. Notably, A6-1 has a relatively long CDRH3, containing 21
aa, which might affect the steric constraints of the DE loop and
thereby sterically disrupt the key step in EV-D68 entry. Our ob-
servations indicate that Abs against EV-D68 that bind to the im-
portant residues R126, I131, and I133, which are the ones spatially
closest to the DE loop, exhibit excellent antiviral effects, but they
do not prove that A6-1 blocks EV-D68 infection by means of
postattachment neutralization. This activity of blocking viral entry
was confirmed in vivo. Mice pretreated with A6-1 were protected
from EV-D68 infection, indicating that A6-1 could be a prophy-
lactic for fighting EV-D68 infection, which is an urgent clinical
need. Meanwhile, our results also show that A6-1 can inhibit EV-
D68 from infecting the brain tissue (Fig. 7B), which shows that the
A6-1 plays some role in preventing the CNS from being infected
with EV-D68. Therefore, we think that A6-1 can be further
evaluated for its activities in other animal models such as acute
flaccid paralysis (37) or asthma induced by EV-D68 infection (52).
The use of this technology to isolate and characterize mAbs induced
by an infection or immunization is feasible, especially in response to
new pathogens. The Ab/virus interaction at structural regions such as the
DE loop might be regarded as a key to designing an efficient EV-D68
vaccine through genetic engineering via reverse vaccinology (53, 54).
Disclosures
The authors have no financial conflicts of interest.
References
1. Messacar, K., T. L. Schreiner, J. A. Maloney, A. Wallace, J. Ludke,
M. S. Oberste, W. A. Nix, C. C. Robinson, M. P. Glodé, M. J. Abzug, and
S. R. Dominguez. 2015. A cluster of acute flaccid paralysis and cranial nerve
dysfunction temporally associated with an outbreak of enterovirus D68 in
children in Colorado, USA. Lancet 385: 1662–1671.
2. Piralla, A., N. Principi, L. Ruggiero, A. Girello, F. Giardina, E. De Sando,
S. Caimmi, S. Bianchini, G. L. Marseglia, G. Lunghi, et al. 2018. Enterovirus-
D68 (EV-D68) in pediatric patients with respiratory infection: the circulation of
a new B3 clade in Italy. J. Clin. Virol. 99–100: 91–96.
3. Xiang, Z., L. Li, L. Ren, L. Guo, Z. Xie, C. Liu, T. Li, M. Luo, G. Paranhos-
Baccalà, W. Xu, and J. Wang. 2017. Seroepidemiology of enterovirus D68 in-
fection in China. Emerg. Microbes Infect. 6: e32.
A NOVEL NEUTRALIZING Ab CAN INHIBIT EV-D68 INFECTION
4. Messacar, K., E. J. Asturias, A. M. Hixon, C. Van Leer-Buter, H. G. M. Niesters,
K. L. Tyler, M. J. Abzug, and S. R. Dominguez. 2018. Enterovirus D68 and acute
flaccid myelitis-evaluating the evidence for causality. Lancet Infect. Dis. 18:
e239–e247.
5. Hellferscee, O., S. Tempia, S. Walaza, E. Variava, H. Dawood, N. Wolter,
S. A. Madhi, M. du Plessis, C. Cohen, and F. K. Treurnicht. 2017. Enterovirus
genotypes among patients with severe acute respiratory illness, influenza-like
illness, and asymptomatic individuals in South Africa, 2012-2014. J. Med. Virol.
89: 1759–1767.
6. Esposito, S., G. Chidini, C. Cinnante, L. Napolitano, A. Giannini, L. Terranova,
H. Niesters, N. Principi, and E. Calderini. 2017. Acute flaccid myelitis associated
with enterovirus-D68 infection in an otherwise healthy child. Virol. J. 14: 4.
7. Engelmann, I., M. Fatoux, M. Lazrek, E. K. Alidjinou, A. Mirand, C. Henquell,
A. Dewilde, and D. Hober. 2017. Enterovirus D68 detection in respiratory
specimens: association with severe disease. J. Med. Virol. 89: 1201–1207.
8. Yu, F., H. Song, Y. Wu, S. Y. Chang, L. Wang, W. Li, B. Hong, S. Xia, C. Wang,
S. Khurana, et al. 2017. A potent germline-like human monoclonal antibody
targets a pH-sensitive epitope on H7N9 influenza hemagglutinin. Cell Host
Microbe 22: 471–483.e5.
9. Robbiani, D. F., L. Bozzacco, J. R. Keeffe, R. Khouri, P. C. Olsen, A. Gazumyan,
D. Schaefer-Babajew, S. Avila-Rios, L. Nogueira, R. Patel, et al. 2017. Recurrent
potent human neutralizing antibodies to Zika virus in Brazil and Mexico. Cell
169: 597–609.e11.
10. Wang, Q., H. Yang, X. Liu, L. Dai, T. Ma, J. Qi, G. Wong, R. Peng, S. Liu, J. Li,
et al. 2016. Molecular determinants of human neutralizing antibodies isolated
from a patient infected with Zika virus. Sci. Transl. Med. 8: 369ra179.
Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021
11. Chen, Z., L. Bao, C. Chen, T. Zou, Y. Xue, F. Li, Q. Lv, S. Gu, X. Gao, S. Cui,
et al. 2017. Human neutralizing monoclonal antibody inhibition of Middle East
respiratory syndrome coronavirus replication in the common marmoset. J. Infect.
Dis. 215: 1807–1815.
12. Demberg, T., E. Brocca-Cofano, P. Xiao, D. Venzon, D. Vargas-Inchaustegui,
E. M. Lee, I. Kalisz, V. S. Kalyanaraman, J. Dipasquale, K. McKinnon, and
M. Robert-Guroff. 2012. Dynamics of memory B-cell populations in blood,
lymph nodes, and bone marrow during antiretroviral therapy and envelope
boosting in simian immunodeficiency virus SIVmac251-infected rhesus ma-
caques. J. Virol. 86: 12591–12604.
13. Sun, M., H. Zheng, Y. Xie, B. Li, H. Long, G. Guo, L. Guo, J. Wang, R. Ning,
Y. Li, and L. Liu. 2017. Functional effector memory T cells contribute to pro-
tection from superinfection with heterologous simian immunodeficiency virus or
simian-human immunodeficiency virus isolates in Chinese rhesus macaques.
Arch. Virol. 162: 1211–1221.
14. Alves, D. A., A. N. Honko, M. G. Kortepeter, M. Sun, J. C. Johnson, L. A. Lugo-
Roman, and L. E. Hensley. 2016. Necrotizing scleritis, conjunctivitis, and other
pathologic findings in the left eye and brain of an ebola virus-infected rhesus
macaque (Macaca mulatta) with apparent recovery and a delayed time of death.
J. Infect. Dis. 213: 57–60.
15. Osuna, C. E., S. Y. Lim, C. Deleage, B. D. Griffin, D. Stein, L. T. Schroeder,
R. W. Omange, K. Best, M. Luo, P. T. Hraber, et al. 2016. Zika viral dynamics
and shedding in rhesus and cynomolgus macaques. [Published erratum appears
in 2017 Nat. Med. 23: 264.] Nat. Med. 22: 1448–1455.
16. de Wit, E., A. L. Rasmussen, D. Falzarano, T. Bushmaker, F. Feldmann,
D. L. Brining, E. R. Fischer, C. Martellaro, A. Okumura, J. Chang, et al. 2013.
Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient
lower respiratory tract infection in rhesus macaques. Proc. Natl. Acad. Sci. USA
110: 16598–16603.
17. Schmitt, K., P. Charlins, M. Veselinovic, L. Kinner-Bibeau, S. Hu, J. Curlin,
L. Remling-Mulder, K. E. Olson, T. Aboellail, and R. Akkina. 2018. Zika viral
infection and neutralizing human antibody response in a BLT humanized mouse
model. Virology 515: 235–242.
18. Zhao, J., R. A. Perera, G. Kayali, D. Meyerholz, S. Perlman, and M. Peiris. 2015.
Passive immunotherapy with dromedary immune serum in an experimental an-
imal model for Middle East respiratory syndrome coronavirus infection. J. Virol.
89: 6117–6120.
19. Johnson, R. F., U. Bagci, L. Keith, X. Tang, D. J. Mollura, L. Zeitlin, J. Qin,
L. Huzella, C. J. Bartos, N. Bohorova, et al. 2016. 3B11-N, a monoclonal an-
tibody against MERS-CoV, reduces lung pathology in rhesus monkeys following
intratracheal inoculation of MERS-CoV Jordan-n3/2012. Virology 490: 49–58.
20. Qiu, X., G. Wong, J. Audet, A. Bello, L. Fernando, J. B. Alimonti, H. Fausther-
Bovendo, H. Wei, J. Aviles, E. Hiatt, et al. 2014. Reversion of advanced Ebola
virus disease in nonhuman primates with ZMapp. Nature 514: 47–53.
21. Liu, Y., J. Sheng, J. Baggen, G. Meng, C. Xiao, H. J. Thibaut, F. J. van
Kuppeveld, and M. G. Rossmann. 2015. Sialic acid-dependent cell entry of
human enterovirus D68. Nat. Commun. 6: 8865.
22. Liu, Y., J. Sheng, A. Fokine, G. Meng, W. H. Shin, F. Long, R. J. Kuhn,
D. Kihara, and M. G. Rossmann. 2015. Structure and inhibition of EV-D68, a
virus that causes respiratory illness in children. Science 347: 71–74.
23. Zheng, H. W., M. Sun, L. Guo, J. J. Wang, J. Song, J. Q. Li, H. Z. Li, R. T. Ning,
Z. N. Yang, H. T. Fan, et al. 2017. Nasal infection of enterovirus D68 leading to
lower respiratory tract pathogenesis in ferrets (Mustela putorius furo). Viruses 9: 104.
24. Meng, W., L. Li, W. Xiong, X. Fan, H. Deng, A. J. Bett, Z. Chen, A. Tang,
K. S. Cox, J. G. Joyce, et al. 2015. Efficient generation of monoclonal antibodies
from single rhesus macaque antibody secreting cells. MAbs 7: 707–718.
25. Ku, Z., X. Ye, J. Shi, X. Wang, Q. Liu, and Z. Huang. 2015. Single neutralizing
monoclonal antibodies targeting the VP1 GH loop of enterovirus 71 inhibit both
virus attachment and internalization during viral entry. J. Virol. 89: 12084–12095.
26. Youssif, C., B. Flix, O. Belbin, and M. Comalada. 2016. Measuring NLR olig-
omerization IV: using Förster resonance energy transfer (FRET)-fluorescence
The Journal of Immunology
lifetime imaging microscopy (FLIM) to determine the close proximity of
inflammasome components. Methods Mol. Biol. 1417: 169–183.
27. Lynch, R. M., P. Wong, L. Tran, S. O’Dell, M. C. Nason, Y. Li, X. Wu, and
J. R. Mascola. 2015. HIV-1 fitness cost associated with escape from the VRC01
class of CD4 binding site neutralizing antibodies. J. Virol. 89: 4201–4213.
28. Arita, M., N. Nagata, T. Sata, T. Miyamura, and H. Shimizu. 2006. Quantitative
analysis of poliomyelitis-like paralysis in mice induced by a poliovirus replicon.
J. Gen. Virol. 87: 3317–3327.
29. Liu, L., H. Zhao, Y. Zhang, J. Wang, Y. Che, C. Dong, X. Zhang, R. Na, H. Shi,
L. Jiang, et al. 2011. Neonatal rhesus monkey is a potential animal model for
studying pathogenesis of EV71 infection. Virology 412: 91–100.
30. Patel, M. C., W. Wang, L. M. Pletneva, S. V. Rajagopala, Y. Tan, T. V. Hartert,
M. S. Boukhvalova, S. N. Vogel, S. R. Das, and J. C. Blanco. 2016. Enterovirus
D-68 infection, prophylaxis, and vaccination in a novel permissive animal
model, the cotton rat (Sigmodon hispidus). PLoS One 11: e0166336.
31. Liao, H. X., M. C. Levesque, A. Nagel, A. Dixon, R. Zhang, E. Walter, R. Parks,
J. Whitesides, D. J. Marshall, K. K. Hwang, et al. 2009. High-throughput iso-
lation of immunoglobulin genes from single human B cells and expression as
monoclonal antibodies. J. Virol. Methods 158: 171–179.
32. Sundling, C., G. Phad, I. Douagi, M. Navis, and G. B. Karlsson Hedestam. 2012.
Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque
B cells. J. Immunol. Methods 386: 85–93.
33. Ichiyoshi, Y., and P. Casali. 1994. Analysis of the structural correlates for an-
tibody polyreactivity by multiple reassortments of chimeric human immuno-
globulin heavy and light chain V segments. J. Exp. Med. 180: 885–895.
34. Day, R. N. 2014. Measuring protein interactions using Förster resonance energy
transfer and fluorescence lifetime imaging microscopy. Methods 66: 200–207.
35. Suzuki, T., C. Miyazaki, A. Ishii-Watabe, M. Tada, K. Sakai-Kato, T. Kawanishi,
and N. Kawasaki. 2015. A fluorescent imaging method for analyzing the bio-
distribution of therapeutic monoclonal antibodies that can distinguish intact
antibodies from their breakdown products. MAbs 7: 759–769.
36. Goedhart, J., and T. W. J. Gadella. 2009. Fluorescence resonance energy transfer
imaging of PKC signalling in living cells using genetically encoded fluorescent
probes. J. R. Soc. Interface 6(Suppl. 1): S27–S34.
37. Imamura, T., M. Okamoto, S. Nakakita, A. Suzuki, M. Saito, R. Tamaki,
S. Lupisan, C. N. Roy, H. Hiramatsu, K. E. Sugawara, et al. 2014. Antigenic and
receptor binding properties of enterovirus 68. J. Virol. 88: 2374–2384.
38. Hixon, A. M., G. Yu, J. S. Leser, S. Yagi, P. Clarke, C. Y. Chiu, and K. L. Tyler.
2017. A mouse model of paralytic myelitis caused by enterovirus D68. PLoS
Pathog. 13: e1006199.
39. Morrey, J. D., H. Wang, B. L. Hurst, K. Zukor, V. Siddharthan, A. J. Van Wettere,
D. G. Sinex, and E. B. Tarbet. 2018. Causation of acute flaccid paralysis by
myelitis and myositis in enterovirus-D68 infected mice deficient in interferon
ab/g receptor deficient mice. Viruses 10: 33.
40. Hixon, A. M., P. Clarke, and K. L. Tyler. 2017. Evaluating treatment efficacy in a
mouse model of enterovirus D68-associated paralytic myelitis. J. Infect. Dis.
216: 1245–1253.
2569
41. Zhang, C., X. Zhang, W. Dai, Q. Liu, P. Xiong, S. Wang, L. Geng, S. Gong, and
Z. Huang. 2018. A mouse model of enterovirus D68 infection for assessment of
the efficacy of inactivated vaccine. Viruses 10: 58.
42. Huang, Y., J. Yu, A. Lanzi, X. Yao, C. D. Andrews, L. Tsai, M. R. Gajjar,
M. Sun, M. S. Seaman, N. N. Padte, and D. D. Ho. 2016. Engineered bispecific
antibodies with exquisite HIV-1-neutralizing activity. Cell 165: 1621–1631.
43. Wu, X., Z. Y. Yang, Y. Li, C. M. Hogerkorp, W. R. Schief, M. S. Seaman,
T. Zhou, S. D. Schmidt, L. Wu, L. Xu, et al. 2010. Rational design of envelope
identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science
329: 856–861.
44. Robbiani, D. F., L. Bozzacco, J. R. Keeffe, R. Khouri, P. C. Olsen, A. Gazumyan,
D. Schaefer-Babajew, S. Avila-Rios, L. Nogueira, R. Patel, et al. 2017. Recurrent
potent human neutralizing antibodies to zika virus in brazil and mexico. Cell
169: 597–609.e11.
45. Neu, U., J. Bauer, and T. Stehle. 2011. Viruses and sialic acids: rules of en-
gagement. Curr. Opin. Struct. Biol. 21: 610–618.
46. Rossmann, M. G., E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith,
H. J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, et al. 1985.
Structure of a human common cold virus and functional relationship to other
picornaviruses. Nature 317: 145–153.
47. Rossmann, M. G. 1989. The canyon hypothesis. Hiding the host cell receptor
attachment site on a viral surface from immune surveillance. J. Biol. Chem. 264:
14587–14590.
48. Smith, T. J., M. J. Kremer, M. Luo, G. Vriend, E. Arnold, G. Kamer,
M. G. Rossmann, M. A. McKinlay, G. D. Diana, and M. J. Otto. 1986. The site
of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating.
Downloaded from http://www.jimmunol.org/ by guest on February 1, 2021
Science 233: 1286–1293.
49. Dang, M., X. Wang, Q. Wang, Y. Wang, J. Lin, Y. Sun, X. Li, L. Zhang, Z. Lou,
J. Wang, and Z. Rao. 2014. Molecular mechanism of SCARB2-mediated at-
tachment and uncoating of EV71. Protein Cell 5: 692–703.
50. Zocher, G., N. Mistry, M. Frank, I. Hähnlein-Schick, J. O. Ekström, N. Arnberg,
and T. Stehle. 2014. A sialic acid binding site in a human picornavirus. PLoS
Pathog. 10: e1004401.
51. Poelman, R., I. Schuffenecker, C. Van Leer-Buter, L. Josset, H. G. Niesters, and
B. Lina, ESCV-ECDC EV-D68 study group. 2015. European surveillance for
enterovirus D68 during the emerging North-American outbreak in 2014. J. Clin.
Virol. 71: 1–9.
52. Itagaki, T., Y. Aoki, Y. Matoba, S. Tanaka, T. Ikeda, K. Mizuta, and
Y. Matsuzaki. 2018. Clinical characteristics of children infected with enterovirus
D68 in an outpatient clinic and the association with bronchial asthma. Infect.
Dis. (Lond.) 50: 303–312.
53. Crowe, J. E., Jr. 2017. Principles of broad and potent antiviral human antibodies:
insights for vaccine design. Cell Host Microbe 22: 193–206.
54. Correia, B. E., J. T. Bates, R. J. Loomis, G. Baneyx, C. Carrico, J. G. Jardine,
P. Rupert, C. Correnti, O. Kalyuzhniy, V. Vittal, et al. 2014. Proof of principle for
epitope-focused vaccine design. Nature 507: 201–206.
The Journal of Immunology 1905

Corrections
Zheng, H., J. Wang, B. Li, L. Guo, H. Li, J. Song, Z. Yang, H. Li, H. Fan, X. Huang, H. Long, C. Cheng, M. Chu, Z. He, W. Yu, J. Li, Y. Gao,
R. Ning, N. Li, J. Yang, Q. Wu, H. Shi, M. Sun and L. Liu. 2018. A novel neutralizing antibody specific to the DE loop of VP1 can inhibit
EV-D68 infection in mice. J. Immunol. 201: 2557–2569.

In Fig. 1E and Fig. 1F, the plots in the second column were incorrect. The correct plots for the second column should have shown the
double positive, CD20+CD27+. The correct x-axis label of the second column should be CD20, and the y-axis should be CD27. The
corrected versions of Fig. 1E and Fig. 1F are shown below. The legend for Fig. 1 was correct as published and is shown below for
reference.

Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

1906 CORRECTIONS

FIGURE 1. The establishment of an EV-D68–infected rhesus monkey model and single memory B cell sorting. Dynamic profiles of EV-D68 in (A) the
blood and (B) the nasal swabs and feces of EV-D68–infected rhesus monkeys. EV-D68 viral RNA extracted from these samples was detected using TaqMan
real-time quantitative PCR. The dotted line represents the SEM of viral load changes in feces; the solid line represents the SEM of viral load changes in
nasal swab. (C) Neutralizing Ab responses to EV-D68 virus after 0, 7, 14, and 28 dpi. The neutralization Ab titer was the reciprocal of the highest serum
dilution that inhibited 50% of the viral CPE. (D) Reactivity of the anti–EV-D68 sera from the infected monkeys against the EV-D68 VP1 protein after 0, 7,
14, and 28 dpi; the absorbance value was measured at 450 nm. The results presented are representative of three independent experiments. (E and F) Single-
cell sorting of EV-D68 VP1 memory B cells by flow cytometry. The numbers in the density plots indicate the percentages of cells gated from the previous
gate side scatter (SSC).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801658