JVI Accepted Manuscript Posted Online 10 June 2015 J. Virol. doi:10.1128/JVI.01295-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza

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A H7N9 Viruses

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Jianmin Wang1, Zhe Chen1, Linlin Bao2, Weijia Zhang1, Ying Xue1, XingHuo Pang3,

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Xi Zhang4, Chuan Qin*2and Qi Jin*1, 5

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1

MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen

Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. 2

Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences

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(CAMS) & Comparative Medicine Center, Peking Union Medical College (PUMC),

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Key Laboratory of Human Disease Comparative Medicine, Ministry of Health,

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Beijing, China.

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Beijing Municipal Center for Disease Control and Prevention, Beijing, China.

4

Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China.

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Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases

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Address correspondence to [email protected] (Q.J.); [email protected]

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(C.Q.).

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J.W., Z.C. and L.B. contributed equally to this work.

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X.P., X.Z., C.Q. and Q.J. contributed equally to this article and are co-senior authors.

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ABSTRACT: H7N9 caused a significant global health concern, due to its severe

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infection and approximately 35% mortality in humans. By screening a Fab antibody

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phage library derived from patients who recovered from H7N9 infections, we

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characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and

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HNIgGH8. The epitope of these two antibodies was dependent on two residues in the

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receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein.

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Both antibodies possessed high neutralizing activity.

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In the spring of 2013, a novel influenza A virus, H7N9, broke out in China (4).

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Influenza A viruses are enveloped RNA viruses in the family Orthomyxoviridae. They

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possess eight negative-sense genomic segments and are classified into subtypes based

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on the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA

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is a trimer of identical subunits. Each HA monomer contains a receptor binding site

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(RBS) in its globular head, involved in virus-receptor recognition and viral invasion

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into cells. NA is a mushroom-shaped tetramer of identical subunits. During

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replication, NA removes sialic acid from cellular glycoproteins and glycolipids and

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from both of the virus glycoproteins and thus enables the virus to be released from the

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host cell. So far 16 HA (H1-H16) subtypes and 9 NA (N1-N9) subtypes have been

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identified from avian natural reservoirs. However, it’s the first time that a H7N9 virus

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being infectious and lethal to humans. Within a few months, the H7N9 had caused

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137 of infection cases, including 45 fatalities (8). Direct contact with infected poultry

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was responsible for most of the human cases (2, 4). Genetic analysis also indicated

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that both HA and NA of the H7N9 influenza virus were closely related to that from

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ducks in China (HA) and from wild birds and ducks from China and Korea (NA) (4,

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6).

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Amantadine and rimantadine, have been widely utilized as anti-influenza drugs.

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Unfortunately, neither was effective against H7N9 infection. Recent studies revealed

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that H7N9 could be inhibited by NA inhibitors, oseltamivir and zanamivir (5).

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Unfortunately, a R292K and a R152K (N9 numbering) mutation that conferred

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resistance to both inhibitors were detected (5, 14). Neutralizing antibodies could

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prevent influenza A virus infection, especially in sporadic human infections (11).

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However, the only neutralizing antibody reported to react with H7 was less effective

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against this recent H7N9 virus (10).

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To screen for human neutralizing antibodies against novel H7N9 virus, we

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established two Fab phage display libraries for λ and κ light chains from the

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peripheral blood mononuclear cells of three recovered patients 3-4 weeks after

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hospitalization, who were infected with H7N9 (A/Anhui/1/2013, -AH1). After three

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rounds of panning with purified H7N9 virions, 816 positive Fab clones and 18 unique

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clones were identified. Among these 18 Fabs, 6 derive from the VH3 family, and 12

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derive from the VH4 family. The light chains of these Fabs are derived from the

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lambda gene family Vλ1 and the kappa gene families Vκ1, Vκ2, and Vκ3. All the 18

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clones demonstrated reactivity with purified virions or viral antigens, while 6 showed

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potent in-vitro neutralizing activity. We then converted these 6 Fabs into full-length

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IgG1 molecules for further study. The results for HNIgGD5 and HNIgGH8 (derived

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from H7N9Fab13 and H7N9Fab14) were reported here. Both Fab clones possessed a

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unique VH4 and Vκ1 sequences (Table 1). There are four amino acid substitutions

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between the two Fabs, with one located in the CDR3 region in the light chain. The

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immunospecificity for HNIgGD5 and HNIgGH8 was further tested by

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immunofluorescence assay (IFA). Both reacted with HA protein but not NA (Fig. 1A).

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A hemagglutination inhibition (HI) assay was then performed. HNIgGD5 and

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HNIgGH8 showed equally strong HI activity, with the HI titer as low as 0.8 μg/ml.

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Next, their neutralizing activity against live H7N9 virus was determined on MDCK

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cells as described before (3). As shown in Fig.1B, both HNIgGD5 and HNIgGH8

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substantially neutralized H7N9 virus on MDCK cells in a dose-dependent manner. To

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determine the epitope regions recognized by the antibodies, escape mutants were

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selected as described previously (3, 13). After five passages, variants were confirmed

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by their similar levels of growth in the presence and absence of the antibody and lack

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of inhibition in HI tests. The entire HA gene was then sequenced. As expected, amino

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acid substitutions were detected. Interestingly, the substitutions occurred at identical

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positions, V186G or L226Q (H3 numbering), both located in the RBS. It has been

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reported that these two amino acids had significant roles in human H7 HA binding

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with human receptor (9, 12). To verify this result, we constructed three HA mutants,

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each had one or both of the two amino acids substituted. The constructs were

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transiently expressed in HEK293T cells and further detected by IFA. As shown in

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Fig.1C, either V186G or L226Q abolished binding of HA with the antibodies. These

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results together demonstrated that the epitope of HNIgGD5 and HNIgGH8 was

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dependent on two residues at positions V186 and L226 of human H7N9 HA. It was

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also in agreement with previous findings that antibodies recognizing the globular head

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had strong HI activity (7). Considering the crucial roles for V186 and L226 in human

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H7 HA binding with the human receptor (9, 12), it was plausible that the antibodies,

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HNIgGD5 and HNIgGH8, bound with the HA protein through the RBS and thus

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interfered with its recognition and interaction with the human cellular receptor.

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In vivo therapeutic efficacy for both antibodies was tested in BALB/c mice. Ten

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mice per group were intraperitoneally injected with 1 or 5 mg/kg of purified HuMAbs

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24 h before the mice were challenged intranasally with 50 μl of a 5×LD50 mouse

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infectious dose of H7N9 virus. Another ten mice immunized with an irrelevant human

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IgG were also infected as control. Mice were observed daily for signs of disease and

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mortality for up to 14 days. As shown in Fig.2A, animals that received an irrelevant

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control antibody succumbed to infection in 5-11 days after viral challenge. In contrast,

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40% of the mice that received 1 mg/kg HNIgGD5 or HNIgGH8 survived, and at 5

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mg/kg the antibodies conferred 100% protection from lethality by H7N9 in the

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infected mice. In addition, mice immunized with an irrelevant control antibody

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rapidly lost weight, compared to mice receiving HNIgGD5 or HNIgGH8 that

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gradually regained body weight at approximately 5 days post-infection (dpi) (Fig. 2B).

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To verify that the protection in the infected mice was due to the inhibition of viral

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proliferation by the antibodies, titers of H7N9 virus in the nose and lungs were

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determined. As shown in Fig.2C, mice that received an irrelevant human IgG had high

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titers of virus in both nose and lungs at 5 dpi. However, after passive immunization

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with either HNIgGD5 or HNIgGH8, viral proliferation was noticeably inhibited and

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virus was difficult to detect in mice treated with 5 mg/kg HNIgGH8 (Fig.2C).

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A total of 454 H7N9 cases resulting in at least 171 deaths were reported by

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September 2014, and most cases were antigenically similar to the -AH1 isolate (1). In

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this study, we characterized two HuMAbs by screening a Fab antibody phage library

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derived from patients who recovered from H7N9 infection. These two antibodies

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showed high neutralizing activity against H7N9-AH1 in vitro and in vivo. At present,

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the neuraminidase inhibitors have still been the main therapeutic antiviral agents

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utilized for treatment of human infections with H7N9. Unfortunately, appearance of

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escape mutants was monitored (5, 14). In this regard, HNIgGD5 and HNIgGH8,

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attacking the virus directly towards the HA protein, thus provide an alternative choice

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for H7N9 treatment. The antibodies are human originated and were originally

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generated in response to the natural infectious H7N9 virus and had protected the

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individual, also powerfully supporting this rational. Epitope identification

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demonstrated that two human receptor preferable residues at V186 and L226 were

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essential for antigen recognition. The essential roles played by the two residues in

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receptor binding limit their variation and consequent antigenic drift. Even escape

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occurred, the antibodies would be suitable to be utilized in adjunction with other

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established methods. At last, considering the possibility for antigen drift caused by

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wide spread of H7N9 influenza in natural reservoirs and the continuing zoonotic

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introductions into humans, more HuMAbs, especially the antibodies targeting distinct

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or more conserved epitopes, are still needed.

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ACKNOWLEDGMENTS

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This study was supported by PUMC Youth Fund and the Fundamental Research

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Funds for the Central Universities (33320140031), the National Natural Science

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Foundation of China (No.31300154), the Chinese Ministry of Science and

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Technology (KJYJ-2013-01-01-01) and Chinese National Major S & T Project

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(2013ZX10004-101). We thank the Shanghai and Beijing Centers for Disease Control

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and Prevention for providing the samples.

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Figure Legends

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Fig.1 HNIgGD5 and HNIgGH8 are able to neutralize H7N9 virus. (A) The

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binding activity of the antibodies to HA and NA proteins were determined by IFA. (B)

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Neutralizing activities of HNIgGD5 and HNIgGH8 against H7N9 virus were tested

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on MDCK cells. An irrelevant human IgG was utilized as control. (C) Epitope

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mapping of the antibodies. The selected amino acids in H7N9 HA were converted to

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the residues as indicated. The mutated HA protein was then transiently expressed in

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cells and detected by IFA.

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Fig.2 In vivo therapeutic efficacy of HNIgGD5 and HNIgGH8 in mice. Mice

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received HNIgGD5 or HNIgGH8 24 h prior to H7N9 infection and were monitored

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daily for 14 days. (A) The survival rate is presented. (B) The average body weight

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change of the mice is shown. (C) Virus titers from nose and lung tissue were

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determined at 5 dpi. Virus titers were substantially reduced in the HuMAb-treated

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groups (p < 0.05, t-test).

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Table

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Table 1. Amino acid sequences of variable regions in the H and L chains of H7N9

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virus-specific Fabs

H7N9Fab13

H7N9Fab14

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a

VR,

VRa

CDR1b

CDR2

CDR3

VH

SGGYYWS

YIYYSGSTDYNPSLKS

GSTGDRHYYYYGMDV

VL

RASQSISSYLN

AASSLQS

QQSYSTPPT

VH

SGGYYWS

YIYYSGSTDYNPSLKS

GSTGDRHYYYYGMDV

VL

RASQSISSYLN

AASSLQS

QQSYSTPST

variable

region;

VH,

V gene segments of the heavy chain;

V gene segments of the light chain. b

CDR, complementarity determining region.

VL,

Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses.

H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibo...
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