Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

Abstract

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.

Full text

Regular paper
Characterization of mAb6-9-1 monoclonal antibody against
hemagglutinin of avian inuenza virus H5N1 and its engineered
derivative, single-chain variable fragment antibody
Róża Sawicka1, Paweł Siedlecki1, Barbara Kalenik1, Jan P. Radomski2, Violetta Sączyńska3,
Anna Porębska3, Bogusław Szewczyk4, Agnieszka Sirko1 and Anna Góra-Sochacka1*
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; 2Interdisciplinary Centre for Mathematical and Computa-
tional Modelling, Warsaw University, Warsaw, Poland; 3Institute of Biotechnology and Antibiotics, Warsaw, Poland; 4Department of Recombinant
Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland
Hemagglutinin (HA), as a major surface antigen of inu-
enza virus, is widely used as a target for production of
neutralizing antibodies. Monoclonal antibody, mAb6-9-1,
directed against HA of highly pathogenic avian inuenza
virus A/swan/Poland/305-135V08/2006(H5N1) was puri-
ed from mouse hybridoma cells culture and character-
ized. The antigenic specicity of mAb6-9-1 was veried
by testing its cross-reactivity with several variants of HA.
The mimotopes recognized by mAb6-9-1 were selected
from two types of phage display peptide libraries. The
comparative structural model of the HA variant used for
antibody generation was developed to further facilitate
epitope mapping. Based on the sequences of the ani-
ty- selected polypeptides and the structural model of HA
the epitope was located to the region near the receptor
binding site (RBS). Such localization of the epitope rec-
ognized by mAb6-9-1 is in concordance with its moder-
ate hemagglutination inhibiting activity and its antigenic
specicity. Additionally, total RNA isolated from the hy-
bridoma cell line secreting mAb6-9-1 was used for ob-
taining two variants of cDNA encoding recombinant sin-
gle-chain variable fragment (scFv) antibody. To ensure
high production level and solubility in bacterial expres-
sion system, the scFv fragments were produced as chi-
meric proteins in fusion with thioredoxin or displayed on
a phage surface after cloning into the phagemid vector.
Specicity and anity of the recombinant soluble and
phage-bound scFv were assayed by suitable variants of
ELISA test. The observed dierences in specicity were
discussed.
Key words: inuenza virus, monoclonal antibody, scFv, hemaggluti-
nin, antigenic epitope
Received: 23 March, 2016; revised: 07 October, 2016; accepted:
14 October, 2016; available on-line: 07 December, 2016
*e-mail: annag@ibb.waw.pl
Abbreviations: CDR, complementarity determining region; HA, he-
magglutinin; HI, hemagglutination inhibition test; HRP, Horseradish
Peroxidase; mAb, monoclonal antibody; PBS, phosphate-buered
saline; pfu, plaque forming unit; scFv, single-chain variable frag-
ment antibody; RBS, receptor binding site; VH, variable region of
the heavy chain; VL, variable region of the light chain
INTRODUCTION
Antibodies are used in many research applications, as
well as in various diagnostic assays and medical treat-
ments. Hybridoma technology enables production of
huge amounts of monoclonal antibodies (mAb) against
the target antigen but it is a laborious, time consuming
and expensive technique. Growing demand for recombi-
nant antibodies and their derivatives prompted the de-
velopment of various expression platforms. Up to date
many attempts of their production in mammalian cells
(Ho et al., 2006), insect cells (Choo et al., 2002), plants
(Dobhal et al., 2013), yeast or bacteria (Rouet et al., 2012)
have been reported. The derivatives of antibodies such
as the antigen-binding fragment (Fab) or the single chain
variable fragment (scFv) are of particular interest. Their
reduced structures and molecular weights facilitate ex-
pression and purication process. The scFv fragment
consists of variable regions of heavy (VH) and light (VL)
chains of immunoglobulin connected by a exible linker
(Kalenik et al., 2014). The scFv can be modied by con-
jugation to drugs or toxins and may penetrate the blood
and tissue more rapidly than a complete antibody (Cao et
al., 2012a; Oriuchi et al., 2005). Easiness of modication
gives many possibilities of direct or indirect detection of
this protein and facilitates manipulations of its activity
(afnity to antigens). These properties are especially im-
portant for such targets as rapidly evolving surface anti-
gens of inuenza virus.
The inuenza viruses belong to Orthomyxoviridae fam-
ily. Their genome consists of a single- stranded negative
sense RNA. Due to the antigenic drift and shift, result-
ing frequently in novel mutants and re-assortants that
are able to cross the species barriers, the inuenza vi-
ruses are still a serious global problem for humans and
their livestock, especially for poultry and pigs. Highly
pathogenic avian H5N1 inuenza viruses are responsible
for serious economic losses to the poultry industry and
have caused numerous threats to public health. Since
1996 when H5N1 virus was isolated for the rst time
in China it has spread rapidly and continued to evolve,
which resulted in a periodic emergence of new phyloge-
netic groups in several regions of the world. Currently,
H5N1 viruses are divided into multiple clades and sub-
clades based on antigenic variation (Smith et al., 2012;
Smith et al., 2015; Li et al., 2013). Hemagglutinin (HA),
a strong surface antigen, is synthesized as a precursor,
HA0 molecule, which is cleaved into two subunits, HA1
and HA2 (Szewczyk et al., 2014). The HA1 subunit en-
compasses the membrane-distal globular head containing
the receptor binding site (RBS) and the vestigial esterase
domain. The HA2 subunit encompasses the stalk region
(Velkov, 2013). The main functions of HA during virus
infection are attachment of the virus to the host cell re-
Epub: No 2016_1292
Vol. 63, 2016
https://doi.org/10.18388/abp.2016_1292

2 2016R. Sawicka and others
ceptor and mediation of the fusion of the virus enve-
lope with the endosomal membrane after endocytosis.
This glycoprotein is a major target for anti-virus antibod-
ies and a subject of high antigenic variability (antigenic
drift). Identication and characterization of its antigenic
epitopes is needed for understanding of the molecu-
lar mechanisms of antigenic drift and viral immune es-
cape, which in turn might result in better selection of
the vaccine strains. The reactivity of selected monoclonal
antibodies with escape mutants of H5 HA were inten-
sively studied but the map of H5 antigenic epitopes is
still incomplete. Most of the characterized mAbs against
HA from H5N1 virus recognize the epitopes localized in
the globular head of HA, most of them overlap the RBS
and are isolate-specic, (e.g. Cao et al., 2012b; Du et al.,
2013; Wu et al., 2014), while antibodies binding to the
stem region have often a broad activity (Tan et al., 2015;
Zuo et al., 2015). Extensive review of H5 HA antigenic
sites was provided by (Velkov et al., 2013) who described
the anti-HA antibodies divided into 3 groups: (i) RBS
selective, (ii) non-RBS, membrane-distal globular domain
selective and (iii) HA2 selective.
The mAb6-9-1 is a monoclonal mouse antibody se-
creted by the hybridoma cell line 6-9-1, which was
generated by us from immune cells of a mouse im-
munized with the recombinant H5 HA from A/swan/
Poland/305-135V08/2006 (H5N1). We described usage
of Fab (antigen-binding fragment) fragment of mAb6-
9-1 in electrochemical immunosensor detecting HA
from H5N1 viruses (Jarocka et al., 2014). It was applied
for detection of HA (17-530 aa) from A/Bar-headed
Goose/Qinghai/12/05(H5N1) and HA1 subunit of HA
from two strains of H5N1 viruses: A/swan/Poland/305-
135V08/2006 and A/Vietnam/1194/2004 (Jarocka et al.,
2014). The strongest binding was observed for the long
HA from A/Bar-headed Goose/Qinghai/12/05(H5N1)
with the detection limit of 2.2 pg/mL. The successful
application of the derivatives of this antibody prompt-
ed us to perform its broaden characterization, especially
mapping of an epitope recognized by mAb6-9-1 on the
structural model of the HA from A/swan/Poland/305-
135V08/2006 (H5N1). Moreover, we described cloning,
expression and characterization of recombinant scFv
fragments derived from the mAb6-9-1. The specicity of
two variants (VLVH and VHVL) of scFv produced in
fusion with thioredoxin were evaluated by indirect and
sandwich ELISA assays and conrmed with the scFv
variant produced by the phage display method.
MATERIALS AND METHODS
Hybridoma propagation and monoclonal antibody
purication. Mouse hybridoma cell line 6-9-1 that se-
creted monoclonal antibody against hemagglutinin from
A/swan/Poland/305-135V08/2006 (H5N1) was gener-
ated from splenocytes obtained after immunization of
mice with the recombinant H5 HA. The hybridoma cells
were maintained in RPMI-1640 medium (20 mM L-glu-
tamine, 10% fetal bovine serum, 5% CO2, 37°C). The
mAb6-9-1 was puried from culture supernatant using
protein G (ThermoScientic).
Comparative modeling of HA from A/swan/
Poland/305-135V08/2006 (H5N1). Structures suitable
for modeling were chosen from a default BLAST search
against the PDB database (accessed February 5th, 2015),
with respect to the serotype (H5), X-ray resolution, and
sequence length. Two structures emerged through these
criteria: 2ibx (A/Vietnam/1194/2004 (H5N1)) and 2fk0
(A/Vietnam/1203/2004 (H5N1)), both having over
90% sequence identity. Preparation of X-ray structures
was done in Chimera (v1.11) and consisted of deletion
of non-standard residues and HOH molecules, N- and
C-terminal x and incomplete side chains repair. Both
structures were aligned with MatchMaker (Meng et al.,
2006) and the most similar chains were used as tem-
plates; 2IBX_A, 2IBX_B, 2FK0_K, 2FK0_L. Modeler
software (Sali & Blundell, 1993) was used throughout
the modeling process. Ten different models were gen-
erated and manually evaluated with respect to hydrogen
bond patterns, AA contact and clashes and Ramachan-
dran plot. The best model was subjected to a minimiza-
tion procedure using AMBER force eld (ff14SB) and
a steepest descent procedure to relieve steric hindrances
(15Å box, TIP3PBOX solvent model and Cl- to neutral-
ize the structure).
Epitopes mapping. Epitope recognition by puri-
ed mAb6-9-1 was determined using two Phage Display
Peptide Libraries, Ph.D. ™-C7C and Ph.D.™-12 (New
England Biolabs) according to the manufacturer’s pro-
tocol (Ph.D Phage Display Libraries, version 1.2, New
England Biolabs). Briey, the 96-well plates (Maxisorb
Nunc) were coated with puried mAb6-9-1 (7.5 µg/
well) and incubated at 4°C overnight. After washing with
TBST (TBS + 0.1% Tween-20) and blocking with BSA
solution (5 µg/ml), the titrated phage peptide library
(Ph.D.™-C7C or Ph.D.™-12) was added to each well
(107–108 plaque forming units (pfu) in 100 µl) and in-
cubated for 60 minutes at room temperature. Unbound
phages were discarded, plates washed 10 times with
TBST and bound phages were eluted with 0.2 M glycine-
HCl, (pH 2.2), 1 mg/ml BSA. In a parallel experiment
the OET protein (see below, Antigens and antibodies
used in ELISA) at concentration 0.8 mg/ml was used
for phage elution. Eluted phages were amplied and
taken to the next panning rounds. After three rounds
of panning several phage clones were randomly select-
ed, subjected to phage ELISA, (performed according to
the protocol described in Ph.D Phage Display Libraries,
New England Biolabs) to conrm their binding capac-
ity with mAb6-9-1. DNA isolated from individual phage
clones was sequenced and amino acid sequence of the
corresponding peptides determined. The Pepitope server
(http://pepitope.tau.ac.il/) (Mayrose et al., 2007a) was
used to map the selected peptides onto the 3D model of
HA from A/swan/Poland/305-135V08/2006 (H5N1).
Using the consensus methodology, both PepSurf (May-
rose et al., 2007b) and Mapitope algorithms (Bublil et al.,
2007) were executed and their results combined into a
single prediction including only residues predicted to be
a part of the epitope in both algorithms. Mapping results
were compared with the surface of the H5 HA structural
model using UCSF Chimera (v1.11).
Synthesis of the scFv cDNA. Primers. Sequences
of primers used in the experiment of scFv synthesis and
cloning are listed in the Supplementary le Table S1 at
www.actabp.pl.
Synthesis of VH and VL regions. Sequences encod-
ing the variable regions of light (VL) and heavy (VH)
chains were obtained according to the method and pro-
cedure described by (Ladiges & Osman, 2001). The main
steps of procedure are described compendiously below.
Total RNA was isolated from the hybridoma cell line
6-9-1 using RNeasy Plus Micro Kit according to the
manufacturer’s instructions (Qiagen). 6.5 µg of isolated
RNA and oligo (dT)15 primer were used to synthesize
blunt-ended cDNA using the cDNA Synthesis System
(Roche). Next, the PCR OG/GO linker was ligated to

Vol. 63 3Monoclonal antibody against hemagglutinin of inuenza virus H5N1
the cDNA by T4 DNA ligase (Invitrogen). The resulting
cDNA was used to amplify VH and VL fragments using
Easy-A High-Fidelity PCR Master Mix (Agilent Technol-
ogies) supplemented with the sets of primers, OG and
IgGkapp or OG and IgG1HC1 for VL and VH synthe-
sis, respectively. IgGkapp primer corresponded to the
constant region of the light chain CL whereas IgG1HCl
corresponded to the CH1 gene (sequences are provided
in Supplementary le 1: Table S1 at www.actabp.pl).The
PCR products were cloned into the pGEM-T easy vec-
tor (Promega). Several clones were sequenced and ana-
lyzed using vbase2 on-line program (http://www.vbase2.
org/vbdnaplot.php) to determine sequences of CDRs of
VH and VL.
Assembling of scFv. Selected functional clones of
light and heavy chain of mouse IgG were used to syn-
thesize scFv construct. First, to obtain scFv in VH-VL
orientation the variable fragments of heavy and light
chains were amplied using two pairs of primers either
VHNdeF and scFvVHR or scFvVKF and VkNotR.
Then VH and VL fragments were assembled into scFv
by overlapping PCR reaction using external primers
VHNdeF and VkNotR. The same method was applied
to synthesize scFv in opposite orientation (VL-VH) us-
ing specic primers: NdeVLF, linkVLR and linkVHF,
VHrevNot. The PCR reactions were performed using
Pfu Polymerase (Fermentas). Finally, PCR products were
digested with NdeI and NotI restriction enzymes and li-
gated into the modied pET26b expression vector (No-
vagen) carrying the sequence encoding His- and Strep-
tags previously cloned by us.
scFv expression in E. coli. The scFv constructs were
re-cloned from the pET26b into the modied pET201
vector carrying the sequence encoding thioredoxin
which resulted in obtaining two recombinant plasmids:
pET201/TVHVL and pET201/TVLVH that were used
for scFv overexpression in E. coli Origami (DE3) strain.
For an efcient production of recombinant TscFvs sev-
eral different conditions of temperature (37°C, 30°C,
25°C, 20°C), IPTG concentration (10 mM, 1 mM,
0.1 mM, 0.05 mM, 0.01 mM) and time of induction (be-
tween 1 h–48 h) were tested. The TscFv expressed by
E. coli were puried on a gravity ow Ni-TED 1000 col-
umn (Marcherey-Nagel) in denaturing conditions accord-
ing to the procedure described in manufacture’s manual
(Marcherey-Nagel). The rst and the second elution frac-
tions were mixed and dialyzed against stepwise decreas-
ing concentration of urea in the range between 6.0–0 M.
Each step lasted at least 3 hours. Purity of recombinant
proteins was evaluated with SDS PAGE.
Phage displayed scFv (PhscFv). The cDNA frag-
ment encoding scFv in VL-VH orientation (without
thioredoxin) was amplied by Easy A polymerase (Easy-
A High-Fidelity PCR Master Mix, Agilent Technologies)
with the RRPvuF and VHrevNot primers (sequences
are provided in Supplementary le 1: Table S1 at www.
actabp.pl) using recombinant plasmid pET26b carrying
scFv cDNA as a template. The obtained PCR prod-
uct was digested with PvuII and NotI and cloned into
pSEX81 phagemid vector (Progen). Small-Scale Phage
Rescue was conducted according to the published proto-
col 154.3.4 by (Dorsam et al., 2002) with minor changes
(rst centrifugation was prolonged to 25 min and 5 h
incubation in 37°C was changed to 18 h in 30°C). The
obtained phages (PhVLVH) were titrated according to
the online protocol ‘Helper Phage Production’ (Stocking-
er Lab website: http://www.oardc.ohio-state.edu/stock-
ingerlab/Protocols/HelperPhage.pdf) and used in Phage
ELISA. The ELISA was conducted according to (Dor-
sam et al., 2002) with minor modications. Briey, Max-
iSorp plates were coated with 500 ng of the antigen and
incubated overnight at 4°C, washed with PBS, blocked
(2 h, 2% skim milk in PBS), incubated with 107-108 pfu
for 2 h, washed and incubated with anti-M13 antibodies
conjugated with HRP (GE Healthcare).
Sequencing of mAb6-9-1. The amino acid sequence
of heavy and light chains of mAb6-9-1 was estimated by
mass spectrometry of trypsin-digested mAb fragments.
The analysis was performed in Mass Spectrometry Lab
at the Institute of Biochemistry and Biophysics Polish
Academy of Sciences, Warsaw.
Hemagglutination inhibition test. HI tests were
performed as described previously (Stachyra et al., 2014;
Stachyra et al., 2016) using inactivated viruses as antigens,
either the highly pathogenic A/Turkey/Poland/35/2007
(H5N1) (National Veterinary Research Institute,
Pulawy, Poland) or the low pathogenic A/chicken/Bel-
gium/150/1999 (H5N2) (DG Deventer, Netherlands).
HI titer was dened as the reciprocal of the highest dilu-
tion of sera that completely inhibited hemagglutination.
Antigens and antibodies used in ELISA. The fol-
lowing antigens corresponding to HA from A/swan/
Poland/305-135V08/2006 (H5N1) were used: (1) HA/
Nde, a short variant containing region of 17-340 aa with
His-tag at N- terminus produced (in our laboratory) in
E. coli; (2) OET, a long variant containing region of 17-
530 aa derived from baculovirus system (Oxford Expres-
sion Technologies, UK); The following recombinant HA
antigens were purchased from Immune Technology Cor-
poration (all of them are 6x His-tagged and derived from
293 cell culture): Qinghai – 17-530 aa from A/Bar-head-
ed Goose/Qinghai/12/05 (H5N1); Vietnam – 17-530 aa
from A/VietNam/1203/2004 (H5N1); Anhui – 18-530
aa from A/Anhui/1/2005 (H5N1); Cambodia – 24–341
aa from A/Cambodia/R0405050/2007 (H5N1); Hubei
– 18-530 aa from A/Hubei/1/2010 (H5N1); Germany
– 17-342 aa from A/Turkey/Germany-MV/R2472/2014
(H5N8); Wuhan – 17-529 aa from A/Wuhan/359-
95(H3N2); Netherlands – 17-527 aa from A/chicken/
Netherlands/1/03 (H7N7); New Caledonia – 18-530 aa
from A/New Caledonia/20/99 (H1N1).
Antibodies. IgY 745 – anty-H5 HA (H5N1) poly-
clonal IgY was puried in our laboratory from eggs
collected from laying hens immunized with DNA vac-
cine based on pCI vector with cloned cDNA sequence
encoding 1-568 aa of HA from A/swan/Poland/305-
135V08/2006 (H5N1) (Stachyra et al., 2014). IgY was
puried using Pierce™ Chicken IgY purication kit
(ThermoFisher). Anti-M13 HRP-conjugated monoclo-
nal antibody (HRP/Anti-M13 Monoclonal Conjugate,
GE Healthcare); anti-Strep-tag II monoclonal antibody
(StrepMAB- Classic, IBA, GmbH); anti – chicken IgY
(Goat anti-Chicken IgY Fc Secondary Antibody, HRP
conjugate, Thermo Scientic); anti-mouse HRP antibody
(Anti-Mouse IgG (γ-chain specic) – Peroxidase anti-
body produced in goat, Sigma Aldrich) were purchased
as indicated.
Indirect and sandwich ELISA. Indirect ELISA.
The 96-well MaxiSorp plates (Nunc, Denmark) were
coated overnight at 4°C with 300 ng of the respective
antigens. Following 4x washing with PBST buffer (0.1 M
PBS with 0.05% Tween-20) plates were blocked with 2%
BSA in PBS for 1.5 h. Next, plates were incubated with
puried antibody (mAb6-9-1 or TscFv) overnight at 4°C
and after washing incubated (1.5 h, 37°C) with the anti-
strep-tag antibody, washed and incubated (1 h, 37°C)
with anti-mouse HRP conjugated IgG. In the case of
mAb6-9-1 the step with anti-strep incubation was omit-

4 2016R. Sawicka and others
ted. Following incubation with TMB (3,3’,5,5’ tetrameth-
ylbenzidine, Sigma-Aldrich) absorbance at 450 nm was
measured. Denatured (reduced) antigens were obtained
after incubation with 10 mM DTT for 1 h at 37°C ac-
cording to (Wu et al., 2014).
Sandwich ELISA. The same plates as in Indirect
ELISA were coated with 500 ng of the TscFv. Wash-
ing and blocking conditions were as above. Next, plates
were incubated with antigens for 1.5 h at room temper-
ature and after washing incubated (1.5 h) with IgY745
anti- H5 polyclonal IgY at various dilutions in the range
1:4 000 to 1:16 000, washed again and incubated (45 min,
room temperature) with HRP-conjugated goat anti-chick-
en IgY Fc (Thermo Scientic) 1:12 000. Following incu-
bation with TMB absorbance at 450 nm was measured.
RESULTS AND DISCUSSION
Prediction of the epitope recognized by mAb6-9-1
In order to facilitate mapping of the antigenic sites
recognized by mAb 6-9-1 the structural model of the
HA variant (A/swan/Poland/305-135V08/2006 (H5N1))
used for antibody generation was created (Fig. 1a) as de-
scribed in Materials and Methods.
Two commercial phage display peptide libraries were
used for selection of peptides that were reactive against
mAb6-9-1. In both of them, the library of random
peptides (linear dodeca- or loop-constrained heptapep-
tides in Ph.D.™-12 Phage Display Peptide Library or
Ph.D.™-C7C Phage Display Peptide Library, respective-
ly) are fused to a minor coat protein pIII of the M13
phage. After three rounds of panning several potentially
positive clones (phage-displayed peptides) were selected
for further analysis. First, they were subjected to Phage
ELISA to verify their binding capacity with mAb6-9-1,
then phage DNA was isolated and sequenced to reveal
the predicted amino acid sequence of the mimotopes
(polypeptides displayed on the surface of the phages).
Phage clones selected from the Ph.D-12 Library were
examined in three concentrations: 109, 108 and 107 pfu/
well, respectively. The absorbance value (OD450) two-
fold higher than the value of the negative control was
considered positive. The rst concentration (109 pfu/
well) was too high, since it resulted in positive signal for
all selected clones. The lower concentrations allowed to
select 7 clones having the highest reactivity among 21
phages examined (Fig. 2). Six of them expressed the
same peptide sequence, VHWDFRQWWQPS, while one
different, FPSDWWSQAWSM.
Similar analyses were performed for phage clones se-
lected from the Ph.D-7C Library and resulted in selec-
tion of four mimotopes (ETDTLTQ, KTFLSSH, PHK-
PAMN and KPYTFVG) which occurred with the similar
frequency.
The Pepitope server was used for epitope mapping
using these afnity-selected peptides and the struc-
tural model of HA from the A/Swan/Poland/305-
135V08/2006 (H5N1). The combined algorithm (con-
sensus results of Mapitope and Pepsurf algorithms) se-
lected one cluster of four HA residues (W138, D140,
F159 and W165) from 6 mimotopes that are shown on
the structural model (Fig. 1c–d). The Pepitope analysis
with only one, (FPSDWWSQAWSM) identied a larger
cluster of 8 HA residues (K135, S136, W138, D140,
A143, V147, S148 and W165) as an epitope. Both clus-
ters shared three residues (W138, D140 and W165). All
of them were localized on the globular head of the HA1
subunit in the proximity of the RBS. Moreover amino
acids W138 and D140 were localized within the anti-
genic epitope called region A, while W165 in the region
B. Antigenic epitopes (A–E) were proposed for H5N1
Figure 1. Model of three-dimensional structure of HA from
A/swan/Poland/305-135V08/2006 (H5N1) (a) and A/Viet-
nam/1194/2004 (H5N1) (b).
Both proteins are represented as Connolly surface, colored by
amino acids hydrophobicity based on Kyte-Doolittle scale (blue
– hydrophilic, red – hydrophobic) (Kyte & Doolittle, 1982). (c) en-
larged head of HA from A/swan/Poland. Yellow surface represents
the six amino acids building the RBS interface (Y107, W165, H195,
E202, L206, and Y207), dark blue depicts the four residues of the
mAb6-9-1 epitope (W138, D140, F159 and W165). W165 is shared
between these two groups and is colored green. (d) ribbon dia-
gram of overlapped structures of the part of HA head from Poland
(cyan color) and Vietnam (pink color). Two residues dening the
binding specicity of mAb6-9-1 are marked on each structure, [1]
D140 (Poland)/S (Vietnam), [2] S145 (Poland)/L (Vietnam).
Figure 2. Reactivity of individual phage clones with mAb6-9-1 in
Phage ELISA.
Plates were coated with mAb6-9-1 or BSA and to each well 108
pfu and 107 pfu were added, plates were read at 450 nm. For
each phage clone signal obtained with target protein (mAb6-9-
1) was compared with that obtained without target protein (e.g.
BSA); presented on the horizontal axis OD value is reduced by the
background (OD value for BSA). For the positive phage clones the
amino acid sequences of the dodecapeptides are provided.

Vol. 63 5Monoclonal antibody against hemagglutinin of inuenza virus H5N1
highly pathogenic avian inuenza viruses based on the
classication of the antigenic domains of H3 (from
H3N2 virus) (Peng et al., 2014). Both epitopes, A and B,
contrary to the epitopes C–E, contribute strongly to the
antigenic variation of the HPAI (highly pathogenic avian
inuenza) H5N1 viruses (Peng et al., 2014).
The conclusions from epitope mapping were veried
by both, the Hemagglutination Inhibition test and ELI-
SA assay. In the HI test ability of mAb6-9-1 to inhibit
binding of H5N1 (by RBS) to receptors on erythrocytes
surface were analyzed. The HI titer was moderately posi-
tive, equal to 32, for the homologous virus, A/Turkey/
Poland/35/2007(H5N1), while for a nonhomologous vi-
rus A/chicken/Belgium/150/1999(H5N2) (DG Deven-
ter, Netherlands) it reached only 8, which was consid-
ered as negative. The HI results suggest that the epitope
recognized by mAb6-9-1 might be localized nearby the
RBS. Similarity between both antigens used in HI test
was 86%, besides HA cleavage sites the differences were
localized around RBS and the mapped epitope (aa posi-
tions: 135, 140, 154, 100, 124, 129, 140, 142, 154, 170,
172, 184, 205, Fig. 3) which may explain the low HI titer
obtained for the nonhomologous antigen.
With ELISA assay we analyzed binding of mAb6-9-1
to various HA antigens from different clades of H5N1
viruses as well as H1, H3 and H7 HA from several se-
lected strains (Fig. 4a). The results showed that mAb6-
9-1 can recognize both, a short (HA/Nde) and a long
(OET) variants of H5 HA from A/swan/Poland/305-
135V08/2006 (H5N1), clade 2.2 as well as H5 HA from
A/Bar-headed Goose/Qinghai/12/05 (H5N1), clade 2.2
and H5 HA from A/Anhui/1/2005 (H5N1), but not the
H5 HA from A/Hubei/1/2010 (H5N1), clade 2.3.2.1,
A/Cambodia/R0405050/2007 (H5N1), clade 1 nor A/
Turkey/Germany- MV/R2472/2014 (H5N8). The data
is in concordance with observation that the most vari-
able inter-clade positions are largely located on the glob-
ular head proximal to the RBS (Velkov et al., 2013). We
also found that mAb6-9-1 does not recognize the H3
HA from A/Wuhan/359/95 (H3N2), the H1 HA from
A/New Caledonia/20/99 (H1N1) nor the H7 HA from
A/chicken/Netherlands/1/03 (H7N7) (Fig. 4a).
Reactivity with HA/Nde antigen containing only the
HA1 part of H5 HA conrmed that the epitope rec-
ognized by mAb6-9-1is located within HA1 subunit
(Fig. 4b). Moreover, ELISA conrmed that mAb6-9-1
binds probably a conformational epitope because dena-
turation of the antigens (reduction of disulde bonds
with 10 mM DTT) resulted in a complete loss of mAb6-
9-1 binding (Fig. 4b).
Comparison of the 3D structures of H5 HA proteins
from A/swan/Poland/305-135V08/2006 (H5N1) and
from A/Vietnam/1194/2004 (H5N1) (Fig. 1a, b and d)
and an alignment of amino acid sequences of the H5
HA antigens tested by ELISA and in HI tests (Fig. 3)
clearly showed that two amino acids, D140 and S145
in the proximity of the predicted epitope might decide
about antigen binding specicity of mAb6-9-1. None of
the analyzed HA proteins with the residue other than as-
partic acid in the position [1] i.e. D140 and other than
serine in the position [2] i.e. S145 was recognized by
mAb6-9-1.
Construction and expression of the scFv – mAb6-9-1
derivative
The cDNA fragments encoding variable regions of
heavy and light chains of mAb6-9-1 were cloned into
the pGEM Teasy vector and sequenced. Each of four
selected clones of VH carried the same (functional) se-
quence. Similarly, one functional sequence was found
among thirty analyzed clones carrying VL, however, ma-
jority of them were classied as nonfunctional due to
an internal stop codon in CDR3. Taking into account
the nature of hybridoma cells such nding is not sur-
prising and was reported previously (Toleikis & Frenzel,
2012). The predicted amino acid sequences of the CDRs
in both variable chains are shown in Table 1. Sequenc-
es encoding VL and VH were assembled in two possi-
ble orientations (VL-VH and VH-VL) with the exible
linker (Gly4Ser)3 into scFv and cloned into the modi-
ed pET201vector downstream the sequence encoding
thioredoxin (Supplementary le Fig. S1 at www.actabp.
pl). The choice of thioredoxin as the fusion partner was
based on the literature data indicating that the formation
of disulde was essential for many scFvs described (Ju-
rado et al., 2006; Sonoda et al., 2010).
Finally, two recombinant variants of scFv in fusion
with thioredoxin (TscFv: TVHVL and TVLVH) were
Table 1 The sequences of complementarity determining regions (CDRs) of scFv6-9-1.
CDR1 CDR2 CDR3
Light chain
Heavy chain
SSVNY
GYSITSDYA
YTS
ISYSGST
QQFTSSPWT
ARSGISYYFGTDY
Amino acids complementarity which determines CDRs of the VH and VL domains were estimated according to the international ImMunoGeneTics
information system® (IMGT, http://www.imgt.org)
Figure 3. Alignment of the fragments of HA antigens in the region adjacent to the mapped epitope of mAb6-9-1.
The residues corresponding to those forming the RBS according to (Al-Majhdi, 2007) and those identied by the Pepitope software
(W138, D140, F159, W165) as the potential target of mAb6-9-1 are marked above the alignment by asterisks and triangles, respectively.
The positions that seem to dene binding specicity of mAb6-9-1 are marked as white letters on a black background and additionally
identied as [1] and [2] below the alignment. Belgium (H5N2) was used only in HI test. For the full names of the viruses refer to the text.

6 2016R. Sawicka and others
Figure 4. Reactivity of mAb6-9-1.
(a) Indirect ELISA with HA antigens in the native form. (b) Indirect ELISA with the selected HA antigens in the native and the reduced
forms. C(-), negative control. The plates were coated with 300 ng of HA from the indicated viruses. Puried mAb6-9-1 and the anti-
mouse IgG conjugated to HRP were used as primary and secondary antibody. The full names of the viruses are provided in the text.
Figure 5. Verication of antigenic specicity of scFv.
(a) Indirect ELISA using TscFv. The plates were coated with 300 ng of the indicated HA antigens or BSA as a negative control, recom-
binant TscFv (TVHVL, TVLVH) were used as primary antibody, while anti-strep IgG served as secondary antibody. (b) Phage ELISA using
phage displayed scFv (PhscFv). Plates were coated with 500 ng of the indicated antigens, incubated with phages (PhVLVH or the nega-
tive control) and with anti-M13 IgG as secondary antibody. (c) and (d) sandwich ELISA using TVHVL and TVLVH, respectively. The plates
were coated with 500 ng of TVHVL or TVLVH, incubated with the indicated HA antigens (250-31 ng/well) or BSA (c-), negative control,
next incubated with anti-H5 HA polyclonal chicken IgY and with anti-chicken IgG-HRP. (e) sandwich ELISA using TVLVH. The plates were
coated with 500 ng/well of TVLVH, concentrations of HA antigens and dilutions of anti-H5 HA polyclonal chicken IgY were as indicated.
C1(-), C2(-) and C3(-) various negative controls: ELISA performed without HA antigen, without IgY 745, and without antigen and IgY745,
respectively.

Vol. 63 7Monoclonal antibody against hemagglutinin of inuenza virus H5N1
produced in E. coli and puried on Ni-TDA agarose.
SDS-PAGE analysis indicated the high purity of TscFvs
obtained and their expected molecular weights to be
about 42 kDa (Supplementary le Fig. S1b at www.act-
abp.pl).
Antigen binding specicity of the recombinant
scFv 6-9-1
Irrespectively of the TscFv variant, in the indi-
rect ELISA the strongest binding was observed with
HA/Nde – short H5 HA (17-340aa) from A/swan/
Poland/305-135V08/2006 (H5N1) (Fig. 5a). Surprisingly,
the TscFv failed to recognize H5 HA from A/Bar-head-
ed Goose/Qinghai/12/05 (H5N1), the protein previous-
ly very well recognized by mAb6-9-1 (Fig. 4a and 5a).
Higher plasticity of scFvs in comparison to the full-
size antibodies may affect their specicity and afnity
(Shepelyakovskaya et al., 2011). To verify if this change
of specicity was due to incorrect scFv folding a phage
displayed variant of scFv (PhVLVH) was obtained. Pub-
lished data showed that the conversion of scFv from
phage-bound to soluble form may results in change of
specicity because the phage pIII protein may provide
structural support for antigen- binding site (Kaku et al.,
2012). The results of phage ELISA (Fig. 5b) showed that
indeed PhVLVH was able to specically recognize the
HA antigen from A/Bar-headed Goose/Qinghai/12/05
(H5N1). These results strongly support the conclusion
that the cloned cDNA encoding the variable regions of
heavy and light chains correspond to the heavy and light
chains of mAb6-9-1. Moreover, comparison of the ami-
no acids sequences determined by mass spectroscopy of
trypsin-digested mAb6-9-1 with CDRs sequences of VH
and VL predicted from nucleotide sequences of cloned
scFv showed 87% and 100% identity, respectively (Sup-
plementary le Fig. S2). The alteration of antigen speci-
city observed by indirect ELISA prompted us to search
for a better method of testing the activity of TscFv.
We performed sandwich ELISA using TscFvs (TVH-
VL and TVLVH, 500ng/well) as capture antibodies, an-
ti-H5 HA IgY 745 as detection antibody and various HA
antigens. TscFvs were capable of targeting not only the
short fragment of H5 HA (HA/Nde) but also the long
fragment of H5 HA from A/Bar-headed Goose/Qing-
hai/12/05 (H5N1) (Fig. 5c–d).
The H5 HA from A/Anhui/1/2005 (H5N1; clade
2.3.4) was also weakly recognized by TscFv, while bind-
ing to H5 HA from A/Vietnam/1203/2004 (H5N1;
clade 1) and to H7 HA from A/chicken/Nether-
lands/1/03 (H7N7) was not detected (Fig. 5c–d). Ob-
tained results showed that binding capacity of TscFv to
the long H5 HA version may be stronger than to the
short H5 HA (HA/Nde) (Fig. 5c–d). This observation
was conrmed in detailed analysis when serial two fold
dilutions of both HA antigens in the range of 250–30
ng/well and anti-H5 HA IgY (1:4 000–1:16 000) were
applied (Fig. 5e). All data from sandwich ELISA were
in accordance with the results obtained for mAb6-9-1
(Fig. 4a).
The observed discrepancies of reactivity of TscFv to-
ward the long version of H5 HA, in two kinds of ELI-
SA, indirect and sandwich (Fig. 5a and 5c, d, e), may
be explained by technical inconsistencies. The lack of
binding to the long antigen was observed in the indirect
assay, when HA antigens were used to coat the plates
and the TscFv bound to the HA antigen was detected
by the anti-Strep antibody. However, the binding with
the same antigen was observed in the sandwich ELISA,
when TscFv was used to coat the plates and HA an-
tigens bound to TscFv were recognized by the specic
polyclonal anti-HA IgY antibody, detected subsequently
by secondary anti-chicken IgY antibody conjugated with
HRP. Due to the high plasticity of scFv, in the indirect
ELISA, the Strep-tag was probably hidden in the confor-
mational structure of TscFv and was not accessible for
the anti-Strep antibody.
In conclusion, epitope recognized by the full-length
monoclonal antibody was mapped in the proximity of
the RBS. This assumption is supported by the results of
HI assay. Unfortunately, contrary to mAb6-9-1, TscFv
did not demonstrate HI activity (not shown). However,
such result does not exclude that scFv recognized the
same epitope as mAb. To prevent hemagglutination, an-
tibody must bind to or near RBS and cause steric hin-
drance (Ascione et al., 2009; Zhang et al., 2013). It is
more likely that the reduced size of scFv is not an effec-
tive barrier between virus and the red cell. Both antibod-
ies display narrow specicity to H5 HA from H5N1 in-
uenza viruses, mostly from clade 2.2 and might be used
effectively in immunosensors specically detecting these
type of viruses (Jarocka et al., 2014; Jarocka et al., 2016).
Acknowledgments
The authors thank Dr. Tomasz Sarnowski (IBB PAN)
for the thioredoxin vector, Agata Malinowska for LC-MS
analysis, and Anna Stachyra, Dr Anna Olszewska, Prof.
Zenon Minta and Prof. Krzysztof Śmietanka for sharing
the results of HI test.
Acknowledgements of nancial support
This work was done in frame of the Polish Vaccine
Consortium and it was supported by EC Innovative
Economy Program, POIG.01.01.02-00-007/08.
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