Accepted Manuscript Title: Generation of monoclonal antibodies reactive against subtype specific conserved B-cell epitopes on haemagglutinin protein of Influenza virus H5N1 Author: Petra Fiebig Awad A. Shehata Uwe G. Liebert PII: DOI: Reference:

S0168-1702(15)00012-X http://dx.doi.org/doi:10.1016/j.virusres.2015.01.006 VIRUS 96508

To appear in:

Virus Research

Received date: Revised date: Accepted date:

22-9-2014 12-12-2014 10-1-2015

Please cite this article as: Fiebig, P., Shehata, A.A., Liebert, U.G.,Generation of monoclonal antibodies reactive against subtype specific conserved B-cell epitopes on haemagglutinin protein of Influenza virus H5N1, Virus Research (2015), http://dx.doi.org/10.1016/j.virusres.2015.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Generation of monoclonal antibodies reactive against subtype specific

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conserved B-cell epitopes on haemagglutinin protein of Influenza virus H5N1

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Petra Fiebiga,1,*, Awad A. Shehataa,b,2, Uwe G. Lieberta,*

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Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt.

E-mail:

Petra

Fiebig:

[email protected],

Awad

A.

Shehata:

[email protected], Uwe Gerd Liebert: [email protected]

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Avian and Rabbit

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Institute of Virology, Leipzig University, Leipzig, Germany;

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* Address correspondence to: Petra Fiebig, [email protected], +49 176 387 367

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49, Windorfer Str. 18, 04229 Leipzig, Germany

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Post-publication also to: Uwe G. Liebert, [email protected], +49 341

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9714301, Institute of Virology, Leipzig University, Johannisallee 30, 04103 Leipzig,

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Germany

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Present address: Petra Fiebig, -

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Omega Diagnostics GmbH, Reinbek, Germany

Present address: Awad A. Shehata, -

Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt.

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Centre for Infectious Medicine, Faculty of Veterinary Medicine, Leipzig University, Germany 1

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Abstract

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H5-specific monoclonal antibodies may serve as valuable tools for rapid diagnosis of

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H5N1 avian influenza virus. Therefore, conserved H5-specific sequences of the

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haemagglutinin (HA) protein were expressed in Pichia pastoris and used for

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generation of monoclonal antibodies (mAbs). The two mAbs, FD6 and HE4, were

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strongly reactive against native HA protein and exhibited specificity for subtype H5.

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By epitope mapping linear epitopes of mAbs were identified that are highly conserved

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among influenza A virus of subtype H5. Additionally no sequence similarities to

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homologous regions on HA proteins of other influenza A virus subtypes (i.e. H1, H3,

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H7, H9) were detected by sequence alignment analysis. Both mAbs did not cross

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react with native or denatured HA proteins of other influenza A virus subtypes.

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Furthermore, using ELISA and immunofluorescence test mAb FD6 reacted only to

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the native H5 protein of recently circulating highly pathogenic H5N1 influenza viruses

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but not to low pathogenic H5N1 isolates. In conclusion, the use of the two mAbs in

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non-molecular tests like antigen-capture-ELISA appears promising for detecting

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influenza A H5N1 virus.

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Keywords: Avian influenza, H5N1, conserved epitopes, monoclonal antibodies,

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Pichia pastoris, ELISA

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1. Introduction Zoonotic Influenza A viruses are endemic in birds and cause sporadic dead-end

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infections in humans. From the known 18 haemagglutinin (H1-H18) and 11

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neuraminidase (N1-N11) subtypes only viruses expressing H1, H2 or H3 and N1 or

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N2 have caused pandemic human respiratory disease like the currently circulated

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H1N1pdm09 (Fouchier et al., 2005, 2005; Sullivan et al., 2010; Tong et al., 2012;

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Tong et al., 2013; Webster et al., 1992, 1992; Webster et al., 2013). However,

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several subtypes of avian influenza A virus e.g. H5N1, H7N7, H9N2, H7N9 and

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H6N1 have been shown to cross the species barrier and infect humans without prior

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adaptation to humans in an alternate host (Claas et al., 1998; Gao et al., 2013;

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Koopmans et al., 2004; Peiris et al., 1999; Wei et al., 2013). Particularly the H5N1

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influenza virus causes high mortality not only in poultry but also in humans. By end of

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June

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(http://www.who.int/influenza/human_animal_interface/HAI_Risk_Assessment/en/,

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last accessed August 3, 2014). Transmission from human to human occurs

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sporadically, usually during care of patients (Ungchusak et al., 2005; Wang et al.,

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2008; WHO, 2008). But as shown in a ferret model only few mutations of the H5N1

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virus are necessary to acquire airborne transmission (Herfst et al., 2012; Imai et al.,

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2012; Russell et al., 2012) and the segmented genome additionally favours

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reassortment and the production of a virus that can cause a human pandemic. These

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findings emphasize the urgent need for improved easy to perform and sensitive

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diagnostic tests for emergency diagnosis to timely initiate anti-viral therapy. As RT-

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PCR requires expertise and technology and antibody detection is not applicable at

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early infection stages, rapid antigen detection is the method of choice. Results are

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provided within short time and can be used at the point of care without sophisticated

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laboratory equipment by untrained personal. However, at present such tests exhibit a

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low sensitivity, detect the highly conserved influenza nucleoprotein and thus do not

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allow influenza virus subtyping (WHO, 2007). New diagnostic tests detecting the viral

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HA-protein could overcome limitations of existing tests when H5 specific capture and

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detector antibodies are used. For generating such antibodies, H5N1 virus or full-

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length HA protein are currently used (Cui and Tong, 2008; He and Kwang, 2013; He

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et al., 2007; Linke et al., 2011; Yang et al., 2009). The influenza HA mediates

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receptor binding and penetration of the virus and is its major antigen. Five

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neutralizing epitopes (site A, B, C, D and E) have been described for HA of subtype

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H3 (Skehel et al., 1984; Wiley et al., 1981). Antigenic characterization of H5 molecule

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using escape mutants of low and high pathogenic H5 strains revealed two antigenic

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sites (Kaverin et al., 2007; Kaverin et al., 2002). Site 1, a loop comprising HA1

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residues 136-141 (H5 numbering here and throughout the text), corresponds to

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antigenic site A of H3 and Ca2 of H1 (Caton et al., 1982). Site 2 is composed of HA1

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residues 151-156 and 182-194 corresponding to site B of H3 and HA1 residues 124-

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129 that correspond to site Sa in the H1 subtype. As these sites represent

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immunodominant regions, they are subject to selective pressure of the host immune

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system. Therefore sequences of the HA protein that are conserved within subtype H5

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and exhibit significant sequence differences to other HA subtypes (e.g. H1, H3, H7)

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might be appropriate candidates to generate monoclonal antibodies (mAbs). Based

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on this assumption, the objectives of this work were to identify such HA regions and

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to generate and characterise mAbs that allow subtyping of influenza viruses with

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specific detection of H5N1.

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2. Materials and Methods 2.1 Cell lines and viruses

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Myeloma cells SP2/0-Ag14, cultured in RPMI 1640 Glutamax I (Gibco, Karlsruhe,

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Germany) supplemented with 10% FCS, 1% (v/v) non-essential amino acids, 1 mM

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sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin

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and Vero cells, cultured in DMEM Glutamax I (Gibco, Karlsruhe, Germany)

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supplemented with 5% FCS (Biochrom AG, Berlin, Germany), were cultured at 37°C

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in a 5% CO2 humidified atmosphere.

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Influenza A virus subtype H5N1 (A/Thailand/1(Kan-1)/2004) was kindly provided by

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Dr. Puthavathana, Mahidol University, Bangkok (Puthavathana et al., 2005). The

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virus was grown on Vero cells and supernatant was harvested 3-4 days post

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infection. All other viruses were obtained as allantoic fluid: provided by Dr.

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Schweiger, Robert Koch Institute, Berlin, Dr. Nieper, Landesuntersuchungsanstalt

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Sachsen, Dr. Truyen, Leipzig University and Dr. Harder, Friedrich Löffler Institut, all

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Germany, and from Dr. Sultan, Avian and Rabbit Diseases Department, Sadat City

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University, Egypt. Table 1 lists influenza virus isolates used in this study.

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To prepare whole virus antigen, clarified supernatant (340 × g, 10 min) was layered

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over a 25% sucrose cushion in NTE buffer (0.1 M NaCl, 10 mM TrisCl pH 7.4, 1 mM

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EDTA) and concentrated by ultracentrifugation (Surespin Rotor, 100,000 × g, 4°C;

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Shaw et al., 2008). The pelleted virus was resuspended in NTE-buffer and tested by

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haemagglutination test. For H5N1 all procedures were performed in BSL-3 facilities.

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2.2 Identification of H5-specific, potential antigenic sites

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Protein sequence of H5 A/Thailand/1(Kan-1)/2004 was analysed for potential B-cell

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epitopes using seven algorithms based on physico-chemical properties (Emini et al.,

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1985; Janin et al., 1978; Karplus and Schulz, 1985; Kolaskar and Tongaonkar, 1990;

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Parker et al., 1986; Pellequer et al., 1993; Ponnuswamy et al., 1980) provided at

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www.imtech.res.in/raghava/bcepred (Saha and Raghava). In general, epitopes had

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to be predicted by at least 3 of the 7 used algorithms, except for exclusively predicted

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epitopes by method of Kolaskar and Tongaonkar (1990). NCBI's Influenza Virus

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Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html, 16.10.2013) was

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used to create HA-protein alignments of 736 H5N1 isolates from 1997-2005 and 206

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H5 strains other than H5N1 from 1980-2006 (Bao et al., 2008). Consensus

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sequences were used for determination of sequence similarities of potential B-cell

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epitopes within subtype H5 using LALIGN Sequence Alignment of ExPASy

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Proteomics Server (http://www.expasy.ch/, 16.05.2014; Gasteiger et al., 2003). The

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same approach was used to determine similarities to human influenza viruses of

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subtype H3N2 (978 isolates, 1990-2006) and H1N1 (252 Isolates, 1990-2006).

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Finally four regions were chosen designated P1, P2, P3 and P4 for generation of

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monoclonal antibodies. Also, chain HA1 (rHA1) and ectodomain of H5 (rH5) were

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cloned and expressed in Pichia pastoris to be used as ELISA antigens.

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2.3 Expression of recombinant peptides

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The HA of influenza virus A/Thailand/1(Kan-1)/2004 virus was cloned in pcDNA3

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plasmid (Invitrogen, Karlsruhe, Germany) and used for amplification of the truncated

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HA-sequences (P1-P4, rHA1) by PCR. Primers used for PCR amplification are

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shown in Table 2. Restriction sites of XhoI (sense primer) and NotI (antisense primer)

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were integrated into these primers for cloning into the expression vector pAOX-α mut,

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as described previously (Shehata et al., 2012). Expression cassettes were

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transformed into Pichia pastoris strains (GS115 and SMDH1186H, Invitrogen,

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Karlsruhe, Germany) using an optimized protocol based on the Pichia Easy Comp™

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Kit (Invitrogen, Karlsruhe, Germany). Positive transformants were selected on zeocin

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containing media and used for expression of recombinant polypeptides fused with a

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C-terminal histidin tag. Transformed yeast cells were grown in shake flasks

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containing yeast peptone glycerol media (YPGly) (1% yeast extract, 2% peptone, 2%

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glycerol). Expression of

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methanol (YPM) pH8 (1% yeast extract, 1% peptone, 2% methanol) while expression

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of P3 and rH5 was induced in buffered methanol-complex medium (BMMY) pH6 (1%

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yeast extract, 1% peptone, 1,34% yeast nitrogen base, 2% methanol) supplemented

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with Pepstatin A (2 µg/ml; Sigma Aldrich, Munich, Germany) when required.

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Supernatants were collected 24 h (P1, P2, P4) or 48 h (P3, rHA1) after induction,

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clarified by centrifugation (1780 × g, 10 min), supplemented with 1 mM

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Phenylmethylsalfonylfluorid

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Pepstatin A. Polypeptides were purified by metal affinity chromatography (IMAC, Ni-

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NTA Agarose, Qiagen, Hilden, Germany) as described previously (Shehata et al.,

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2012). Expression and purification was verified by western blot using (His)6-tag-

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antibody (1:200, Dianova, Hamburg, Germany). The concentration of purified

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polypeptides was measured using Biorad protein assay kit (Biorad, Hercules, CA,

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USA).

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P1, P2, P4 and rHA1 was induced in yeast peptone

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2.4 Generation of monoclonal antibodies

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Balb/c mice were immunized subcutaneously using 50 µg (in 50 µl PBS) recombinant

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polypeptide mixed with an equal volume of Gerbu Adjuvant 10 (Gerbu Biochemicals

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GmbH, Schwabach, Germany) and boosted with identical doses 2 and 3 weeks

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thereafter. Additional three boosts of 25 µg polypeptide in PBS were given on day 3,

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2 and 1 before mice were sacrificed. Spleen cells were harvested and fused with

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SP2/0-Ag14 cells at 2:1 ratio using polyethylene glycol (PEG) 1500 (Roche

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Diagnostics GmbH, Mannheim, Germany). Hybridoma cells were seeded into 96-well

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plates and selected in hypoxanthine-aminopterin-thymidine (HAT) supplemented

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RPMI 1640 Glutamax I medium (Sigma-Aldrich Chemie GmbH, Munich, Germany).

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After cultivation for 3 weeks aminopterin was omitted from medium and supernatants

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were screened for antibody reactivity and specificity by ELISA. Cells of positive

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tested wells were subcloned by limiting dilution method and screened again. MAbs

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were purified from supernatant using T-Gel Absorbent® (Pierce, Schwerte,

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Germany). Isotyping was performed using a mouse Immunglobulin Isotyping ELISA

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Kit (BD Pharmingen™, Heidelberg, Germany) according to the manufacturer.

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Determination of antibody concentration was performed with Bradford assay.

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2.5 Indirect ELISA

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For indirect ELISA, homologous recombinant polypeptides (1 µg/ml) used in mouse

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vaccination or whole virus antigens (20 HAU/ml) diluted in carbonate buffer (pH 9.6)

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were coated on Microlon high binding microtiter plates (Greiner Bio-One GmbH,

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Frickenhausen, Germany) and incubated overnight at 4 °C. ELISA was performed as

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described previously (Shehata et al., 2012). Briefly, plates were blocked with

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3% BSA in PBS (PBSA) containing 0.05% Tween 20 and hybridoma supernatant or

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purified mAb diluted in blocking buffer was added (2 h, 37°C). After addition of

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horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin (diluted

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1:2000, Dako Cytomation GmbH, Hamburg, Germany) for 90 min, 37 °C and

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3,3′,5,5′tetramethylbenzidin (TMB) substrate, colour development was stopped after

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30 min with 1 N sulphuric acid and absorbance was measured at 450/650 nm. Plates

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were washed 5 times with PBS containing 0.05% Tween 20 after each incubation

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step. Antibody titers of mice sera were expressed as endpoint titer and the cutoff was

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determined using sera of mice immunized with PBS plus adjuvant (Frey et al., 1998).

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2.6 Western blot and Epitope Mapping

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For Western blot analysis, recombinant peptides (1 µg/lane) and whole virus antigens

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(4 HAU/lane) of different HA subtypes were separated by SDS-PAGE and transferred

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onto PVDF membrane (Roti-PVDF, Roth, Nuremberg, Germany). For Epitope

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Mapping fifteen amino acids long peptides overlapping by 13 amino acid residues

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covering the sequences of P1, P2, P4, and rHA1, respectively, were spotted onto a

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cellulose membrane (IZKF-Leipzig, Leipzig, Germany). Membranes were blocked

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with 4% skim milk in PBS and incubated with hybridoma supernatant overnight at

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4 °C, followed by incubation with HRP-conjugated rabbit anti-mouse immunoglobulin

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(Dako, 1:2000) for 90 min at room temperature. Detection was done using

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chemiluminescence. Membranes were washed 3 times for 10 min with PBS after

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each incubation step.

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2.6 Immunofluorescence assay

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Vero cells (grown on glass cover slips) were infected with Influenza virus at

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multiplicity of infection (m.o.i.) of 0.01. After 1 h incubation the supernatant was

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replaced by DMEM supplemented with 5% FCS for viruses exhibiting a multibasic 9

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HA-cleavage site or 2 µg/ml L-1-tosylamide-2-phenylethyl chloromethyl ketone

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(TPCK)-treated trypsin (Sigma-Aldrich Chemie GmbH, Munich, Germany) and 0.2%

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BSA for viruses with a mono basic HA-cleavage site. After 24 h cells were fixed with

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80% ice cold acetone in water at -20°C for 30 min. Cells were blocked with 5% PBSA

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for 30 min at 37°C in a humidified chamber. Hybridoma supernatant was added and

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incubated for 90 min, 37°C. FITC conjugated goat anti-mouse immunoglobulin (Dako,

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1:100) was added to cells and incubated for 45 min. Cover slips were washed

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between each step with 0.5% PBSA. After mounting cover slips onto a glass slide

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using mounting fluid (10% glycerine in PBS) cells were screened for fluorescent

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staining.

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2.7 Homology analysis and cross-reaction examination

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Identified epitopes were analysed for similarities within H1-, H3-, H7- , H9- and H5-

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subtypes using „Identify Short Peptides in Proteins“ provided by Influenza Research

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Database (IRD, http://www.fludb.org, 16.05.2014, Squires et al., 2007). Isolates from

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1990-2013 with complete HA protein sequences were used for sequence analysis

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exclusively. Doubling sequences of one isolate were excluded as well as viruses

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obtained by genetic manipulations.

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2.8 Neutralization assay

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The neutralizing activity of mouse immune sera as well as mAb were tested by

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neutralization test according to the WHO manual (WHO, 2002). Briefly, 50 µl mouse

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serum or mAb (25 µg/50 µl) were mixed with an equal volume of virus H5N1

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containing 100 TCID50/50 µl and incubated at 37°C. After 1 h the mixture (100 µl/well)

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was transferred to a flat-bottomed microtiter plate with a Vero cell monolayer (2×106

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cells/plate). After 1 h incubation at 37°C, supernatant was removed and 200 μl of the

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DMEM containing 5% FCS was added and incubated at 37°C in a 5% CO2 humidified

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incubator. After 3 days of incubation, plates were read as positive or negative

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according to presence or absence of cytopathic effect.

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2.9 Haemagglutination assay and haemagglutination inhibition assay

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Haemagglutination assay for virus titration and haemagglutination inhibition (HI) for

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examination of mouse sera and mAbs were performed according to the OIE manual

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of diagnostic tests and vaccines for terrestrial animals (World Organisation for Animal

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Health (OIE), 2004).

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2.10 Antigen capture ELISA for detection of influenza A virus subtype H5 in

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clinical samples

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Working concentrations of mAbs were determined by checkerboard titration and

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comparison of highest signal-to-noise ratio for the detection of H5 and non-H5

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subtypes. Different concentrations (1 and 2 µg/ml) of mAb FD6 (IgM) or HE4 (IgG)

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were coated as capture antibody in 50 µl carbonate buffer on Microlon high binding

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microtiter plates (Greiner Bio-One) and incubated overnight at 4 °C. After removal of

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unbound antibody, plates were blocked with 3% PBSA 0.05% Tween 20 for 1 h.

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Afterwards 5 HAU of Influenza virus isolates were added at volumes of 75 µl/well and

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incubated for 2 hrs. Each sample was tested in duplicate. Detection of bound antigen

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was carried out by addition of 100 µl/well non-conjugated detection antibody HE4 or

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FD6, respectively for 90 min, followed by addition of HRP-conjugated heavy chain

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specific antibody (Dianova, goat anti-mouse IgG1 or goat anti-mouse IgM,

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respectively, both diluted 1:5000) for 90 min.

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Thirty tracheal swabs obtained from commercial layer chickens in Egypt were

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assessed. Chickens were suspicious for infection with avian influenza. The clinical 11

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symptoms consisted of severe respiratory manifestations, petechial haemorrhages

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on the shank and severe congestion of internal organs. Six influenza negative

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tracheal swabs were obtained from healthy chickens to be used as negative control.

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For detection of H5N1 in clinical samples (tracheal swabs from animals with clinical

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disease and from healthy controls), mAb FD6 (IgM) was used as capture antibody

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(2 µg/ml) and mAb HE4 (2 µg/ml) was used as detector antibody. The cut-off value

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was defined as mean OD450 of negative controls + 3*SD.

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2.11 RT-PCR

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H5N1 infection of chickens was verified by RT-PCR, for which RNA was extracted

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from specimens using Viral Gene-spin™Viral DNA/RNA Extraction kit (Intron

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Biotechnology, Freiburg, Germany) according to manufacturer’s protocol. Reverse

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transcription of H5 and N1 gene was performed using AMV Reverse Transcriptase kit

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(Promega, Mannheim, Germany) with CU-H5F and CU-N1F primers (Barbazan et al.,

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2008), respectively. To amplify a 189 bp fragment of H5 and 131 bp fragment of N1

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primer pair of CU-H5F/CU-H5R or CU-N1F/CU-N1R (Barbazan et al., 2008) were

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used, respectively. The PCR-program was as follow: 95°C 2 min, 35 × (95°C, 30

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sec; 55°C, 45 sec; 72°C, 1min) 72°C 10 min. PCR products were analysed by 1%

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(w/v) agarose gel electrophoresis.

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3. Results 3.1 Identification of potential H5-specific antigenic sites

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Out of 20 predicted potential epitopes four regions, designated P1, P2, P3 and P4

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(Table 3 and Figure 1) were eventually chosen that comprises epitopes with

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conserved sequences within subtype H5 but sequence differences to other subtypes.

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Additionally, these epitopes have not been described for Influenza H5 in literature.

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Polypeptide P1 comprises aa 43-82 and corresponds in its location to the neutralising

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epitope site E of H3 subtype (Skehel et al., 1984; Wiley et al., 1981). Glycosylated

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polypeptide P2 (aa 165-261) encompasses the receptor binding site and parts of

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identified site B epitope of H3, also described for H5 (site 2, Kaverin et al., 2002). P3

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comprises the C-terminus of chain HA1 (aa 292-330), which is unique for H5 protein

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of highly pathogenic H5N1 viruses, due to multiple basic amino acid residues at the

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cleavage site. Polypeptide P4 is located within HA2 moiety (aa 38-132), which is

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highly conserved among different influenza A virus subtypes.

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3.2 Production of mAbs

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Secretory expression of polypeptides P1-P4 in Pichia pastoris was verified by

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Western blot using (His)6-tag-antibody (Figure 2). Purified peptides were used for

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immunization of Balb/c mice. Polypeptides P1, P2 and P4 but not P3 induced

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seroconversion. Serum antibodies against P1, P2 and P4 reacted with homologous

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antigens (polypeptide used for immunisation) and with whole H5N1 virus antigen in

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ELISA, although with 50-100-fold lower endpoint titers. No cross reactivity was

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observed with viruses of subtype H3 and H1 using ELISA (data not shown). Sera

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obtained from P1, P2, and P4 vaccinated mice reacted only with H5 viruses based on

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IFA performed on Vero cells infected with different influenza virus subtypes (Figure

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3). No neutralisation or inhibition of haemagglutination was observed for any of the

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sera (data not shown).

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After somatic cell hybridisation of mouse spleen cells with myeloma cells several

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hybridoma cell lines were selected by HAT supplemented medium. Screening by

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ELISA on homologous antigens and whole virus H5N1 revealed two monoclonal cell

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lines, designated HE4 and FD6 that produced mAbs with reactivity and specificity to

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H5N1 virus.

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3.3 Characterisation of mAb FD6 and HE4

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In Western blot using recombinant polypeptides or whole virus as antigens, mAb FD6

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(isotype IgM) reacted specifically with polypeptides comprising region P1 and viruses

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of subtype H5 (Figure 4A). Similar reactions were detected when mAb FD6 was used

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as primary antibody for ELISA, although it failed to react with H5N6-virus coated with

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a concentration of 1 HAU/well (Figure 4C). The mAb HE4 (isotype IgG1) reacted

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specifically with recombinant peptides comprising region P2 (i.e. P2, rHA1) and

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viruses of subtype H5 using Western blot (Figure 4B) and ELISA (Figure 4C). Both

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mAbs did not react with influenza viruses of other subtypes i.e. H3 and H1. IFA was

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performed on Vero cells infected with H5N1 and influenza virus of other subtypes to

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determine the ability of mAbs to recognise conformational haemagglutinin. MAb HE4

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and mAb FD6 showed a specific staining of H5N1 virus infected cells with low

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background (Figure 4D). As observed with ELISA, mAb FD6 did not recognise H5N6

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infected cells, although it reacted with denatured H5N6 virus in Western blot.

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Both mAbs failed to neutralise or inhibit H5N1 virus (data not shown).

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Immunoblotting using the membrane with spotted peptides covering P1-sequence

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indicated that the sequence VKPLILRDCSV is the linear epitope of mAb FD6. Using

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the membrane with spotted peptides covering HA1-sequence, minimal amino acid

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sequence required for mAb FD6 binding was determined as VKPLI (data not shown).

324

For mAb HE4, the sequence QEDLLVLWGIH was identified as linear epitope using

325

the membrane with spotted peptides covering sequence of P2 and rHA1. Amino acid

326

residues at the N and C terminus, i.e. QE as well as IH, are the essential

327

components of the epitope binding site. No antibody binding was observed with

328

peptides missing residues QE or IH.

329

3.4 Homology and cross-reactivity analysis

330

Using „Identify Short Peptides in Proteins“, isolates of H1, H3, H7, H9 and H5

331

subtypes were analysed for similarities with determined epitope sequence. Of the

332

1537 analysed H5N1 isolates 94% exhibited the epitope sequence of mAb FD6,

333

while 49% of 461 analysed H5 isolates (other than H5N1) comprised the sequence.

334

But it was not found within the H1, H3, H7 or H9 sequence (Table 4). Analysis of 11

335

amino acid long epitope of HE4 revealed no exact matches in H1 or H3 protein

336

sequence, but 29.6% of H3Nx isolates and 90% of H1Nx isolates held a sequence

337

similarity higher than 50%. But all the detected similarities were located between the

338

identified anchor amino acids QE and IH (Figure 5). Looking for identities in anchor

339

amino acids only, no consensus was found in H3 and H1 isolates. Of the 574

340

analysed H7Nx isolates only 1.9% and 17.7% of the 937 H9Nx isolates exhibit a

341

sequence similarity of 55% to the epitope of mAb HE4. But similarities are mainly

342

found in the C-terminal epitope region, including anchor amino acids IH. But as N-

343

terminal anchor amino acids QE are not present a binding of the mAb to H7 or H9 is

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not expected. However, 92% of the 1537 analysed H5N1 isolates and 39.9% of 461

345

H5 isolates other than H5N1 contain the anchor amino acids (Table 4).

346

3.5 Antigen Capture ELISA (AC-ELISA)

347

The optimal antibody concentrations was determined as 100 ng/well for FD6 (capture

348

antibody) and 100 ng/well mAb HE4 (detector antibody) in conjunction with HRP-

349

conjugated Fcɣ1-specific antibody. Signal-to-noise ratios indicated that H5N1

350

samples are distinguishable from samples containing H3N8, H1N1 and H5N6 (5 HAU

351

each). Interestingly, the AC-ELISA did not work when mAb HE4 was used as capture

352

and FD6 as detector in conjunction with HRP-conjugated µ-chain specific antibody.

353

This might indicate a degradation of the IgM antibody within its Fc-part.

354

Optimized conditions using FD6 as capture antibody were used to analyse analytical

355

specificity of the test. It turned out that HPAIV H5N1 from Thailand and Egypt are

356

detected specifically while other HA-subtypes like H1 and H3 were not detected

357

(Figure 6A) even at high concentrations of 1500 HAU (data not shown). Additionally,

358

other H5-subtypes like H5N3, H5N2, low pathogenic H5N1 and outgrouped high

359

pathogenic H5N1 from 1959 are not detected, even at high concentrations. Subtype

360

H5N6 on the other hand is detected in a concentration dependent manner (Figure

361

6B). Compared to H5N1 50 HAU of H5N6 are needed to obtain values above OD450

362

0.2. These concentrations are usually not obtained in specimens, thus AC-ELISA is

363

specific for H5N1, which can be detected even at low concentrations of 2 HAU.

364

To verify applicability of AC-ELISA, thirty specimens derived from chickens showing

365

clinical manifestations of H5N1 infection were tested by AC-ELISA and RT-PCR in

366

parallel. By AC-ELISA 13 out of 30 specimens were tested positive, whereas 16

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samples were tested RT-PCR positive. In comparison to RT-PCR the AC-ELISA

368

exhibits 81% relative sensitivity and 100% relative specificity (Table 5).

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369

4. Discussion In the present work two mAbs were generated that are specific for H5 Influenza virus

371

antigen and allow its specific detection in an antigen-capture ELISA. As the

372

emergence of new pandemic influenza viruses of different HA subtypes is a

373

continuous threat, easy-to-handle rapid antigen detection tests for point of care

374

testing are required that allow viral subtyping. Therefore, HA subtype specific

375

monoclonal antibodies are advantageous. The HA, a type I membrane protein,

376

comprises the major antigen of influenza A virus but its immunodominant epitopes

377

underlie continuous mutations resulting in viral escape from host immune detection

378

(Laursen and Wilson, 2013). Due to this immunodominant regions should not be

379

targets of diagnostic antibodies, as they might loose affinity. Accordingly, in this study

380

two mAbs were generated which target immune-subdominant epitopes. These are

381

not overlapping with known neutralising epitopes and were identified by analysis of

382

the H5 protein with algorithms for prediction of linear B-cell epitopes. Sequence

383

conservation within subtype H5 and differences to other subtypes were also

384

important epitope characteristics. The pre-identificaiton of potential H5-specific

385

epitopes allows the recombinant expression of defined HA protein regions and the

386

directed production of antibodies towards these epitopes, which reduces extensive

387

selection procedures in the aftermath necessary when whole virus is used as

388

immunisation antigen (Linke et al., 2011). MAb FD6 (IgM) and mAb HE4 (IgG1) were

389

obtained by immunisation of mice with recombinant peptides P1 and P2, followed by

390

somatic cell hybridisation and selection, respectively. Both antibodies bind to

391

denatured whole virus of subtype H5 as verified by Western Blot but not to other

392

influenza virus subtypes. While mAb HE4 binds also to native forms of the H5 protein

393

of all H5 isolates, the specificity of FD6 is restricted to recent high pathogenic H5N1

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strains. It reacted not with low pathogenic H5N1, the outgrouped H5N1 or other H5

395

subtypes. The difference in recognition by mAb FD6 might be explained by the

396

recognised epitope and the HA protein sequences. By epitope mapping the linear

397

epitope (DLDG)VKPLILRDCSVAGW (residues 43-60 in H5 protein) with a sequence

398

minimum of VKPLI was identified for mAb FD6. Sequence analysis of HA protein

399

from H5N1 and H5N6 revealed amino acid substitutions D43S and D45N in H5N6,

400

which might influence conformation of the protein and therefore epitope accessibility.

401

Thus mAb FD6 seems to distinguish between H5N1 and other H5 subtypes.

402

Sequence analysis indicates that 98.3% of H5Nx isolates (x = subtype 2-10, 461

403

isolates) misses DLDGVKPLIL, with mutations predominantly within D43, L44 and

404

D45. On the other hand, the vast majority of analysed H5N1 isolates (n=1537) carry

405

the identical sequence with maximum one mutation at residue 43 or 45 (91.5%).

406

Experiments using such strains like A/Hong Kong/485/97 are necessary to confirm

407

antibody binding to native HA in spite of one mutated residue.

408

Epitope of mAb HE4 has been identified as the N-terminal residues 5-15

409

QEDLLVLWGIH of peptide P2, which corresponds to residues 165-179 in H5 protein

410

and thus lies between the two sites comprising the discontinuous epitope site 2

411

(Kaverin et al., 2002). Epitope analysis of mAb HE4 revealed terminal residues QE

412

and IH as important anchor sites for antibody binding, whereas amino acid residues

413

in between are not important. This finding was supported by ELISA, Western Blot and

414

IFA where H1N1 and H3N2 strains were not detected by mAb HE4 although

415

homologous epitope region of H3 protein KFDKLYIWGVH has a similarity of 55% to

416

epitope of mAb HE4. Similar results were found for homologous region of H1 protein

417

(EKEVLVLWGVH). As only a minority of the avian influenza H7 and H9 proteins

418

comprise the C-terminal anchor amino acids, but all isolates miss the N-terminal

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anchor amino acids a binding of mAb HE4 is not expected. As IgM antibody FD6 has

420

a lower avidity to its antigen (data not shown) but in a competitive ELISA it is not

421

competed by HE4, which suggests that binding of one antibody is not blocked by the

422

other (data not shown) and they can be used as capture and detector antibody in an

423

AC-ELISA. Additionally, due to the selective epitope binding of mAB FD6 to

424

conformational haemagglutinin the AC-ELISA allows the discrimination of recent

425

HPAIV H5N1 to low pathogenic H5N1 and other H5Nx subtypes. With a reproducible

426

detection limit of 2 HAU this mAb combination is not more sensitive than other

427

available test (general sensitivity of >1 HAU). In contrast Linke and co-workers

428

generated a test with a detection limit of 0.04-0.05 HAU. Besides the combination of

429

two mAbs for detection and or capture, the use of Poly-HRP40 conjugate improved

430

the detection limit significantly (Linke et al., 2011). The described two mAbs were

431

used singular as detector or capture antibody and they were not HRP conjugated.

432

Instead bound capture antibody was verified by heavy chain specific HRP-conjugated

433

antibody. This resulted in relatively high background, which did not allow detection of

434

lower antigen amounts i.e. 1 HAU. By conjugation the high background might be

435

overcome and detection limit improved.

436

To verify practicability of the AC-ELISA with field samples, 30 tracheal swabs from

437

commercial layer chickens were comparatively analysed by AC-ELISA and RT-PCR.

438

The AC-ELISA had a specificity of 100% but a somewhat reduced sensitivity,

439

particularly with samples of low viral load. Conjugation of detector antibody HE4 to

440

HRP or biotin might improve the performance.

441

Although no monoclonal antibodies were generated, immunisation of Balb/c mice

442

with P4 resulted in H5-subtype specific serum antibodies, which did not cross-react

443

with HA subtypes H3 and H1. This renders the approach promising for generation of

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subtype specific antibodies binding within HA2 moiety, which contributes less to the

445

antigenicity of HA (Vareckova et al., 2003) and therefore should acquire fewer

446

mutations that interfere antibody binding.

447

Somewhat unexpected was the low immunogenicity in inbred Balb/c mice of P3 as

448

the conformation of peptides derived from N- or C-termini of proteins exhibit more

449

likely a conformation similar to this within the native protein and therefore should

450

induce suitable antibodies (Hanly et al., 1995).

451

In conclusion, this paper describes the generation of H5-specific monoclonal

452

antibodies with high specificity and their use in an AC-ELISA. Their use in

453

combination with other described H5 specific antibodies or with commercial available

454

test for nucleoprotein detection may help to create a highly H5-specific and sensitive

455

test system for influenza virus diagnostic without sophisticated technologies.

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456 457

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WHO, 2007. Recommendations and laboratory procedures for detection of avian

619

influenza A(H5N1) virus in specimens from suspected human cases.

620

http://www.who.int/influenza/resources/documents/h5n1_laboratory_procedures/e

621

n/. Accessed 4 November 2013.

622 623

WHO, 2008. Update on Avian Influenza A (H5N1) Virus Infection in Humans. N Engl J Med 358 (3), 261–273.

624

Wiley, D.C., Wilson, I.A., Skehel, J.J., 1981. Structural identification of the antibody-

625

binding sites of Hong Kong influenza haemagglutinin and their involvement in

626

antigenic variation. Nature 289 (5796), 373–378. 28

Page 28 of 43

627

World Organisation for Animal Health (OIE), 2004. Manual of Diagnostic Tests and

628

Vaccines for Terrestrial Animals, Chapter 2.3.4. Avian Influenza, version adopted

629

may 2012. www.oie.int/international-standard-setting/terrestrial-manual/access-

630

online/. Accessed 4 November 2013. Yang, M., Clavijo, A., Graham, J., Salo, T., Hole, K., Berhane, Y., 2009. Production

ip t

631

and diagnostic application of monoclonal antibodies against influenza virus H5. J

633

Virol Methods 162 (1-2), 194–202.

Ac ce pt e

d

M

an

us

cr

632

29

Page 29 of 43

Legends to Figures

635

Figure 1: Diagram of influenza A virus HA protein. Truncated sequences (P1, P2,

636

P3, and P4 as well as recombinant rHA1) and their position within the linear protein

637

sequence are shown. Arrow indicates cleavage site between HA1 and HA2, which

638

remain connected via a disulfide bond.

ip t

634

639

Figure 2: Western blot of purified polypeptides using (His)6-tag-antibody. The

641

theoretical molecular weights of P1, P2, P3 and P4 are 7.59 kDa, 16.06 kDa, 7.67

642

kDa and 14.26 kDa, respectively.

us

cr

640

an

643

Figure 3: Immunofluorescence assay using murine immune sera (dilution 1:50)

645

on Vero cells infected with influenza virus. As control monoclonal Influenza-A

646

nucleoprotein-specific antibody (anti-NP, kindly provided by Prof. Jassoy, Institute of

647

Virology, Leipzig University) was used. No reactivity was seen with P3 immune

648

serum.

d

Ac ce pt e

649

M

644

650

Figure 4: Reactivity of mAb FD6 and HE4. Using Western blot mAb FD6 (A) and

651

HE4 (B) reacted only with H5 viruses. Indirect ELISA (C) and IFA (D) revealed

652

reactivity of both mAbs with H5N1, mAb HE4 reacted additionally with H5N6.

653 654

Figure 5: Sequence alignment of identified linear epitopes (shaded in grey) with

655

adjoining residues and the homologous regions of other influenza virus

656

subtypes. Identical residues are shown by dots. Sequence conservation is detected

657

within H5 subtype for both epitopes, while sequence differences are found in H1, H2, 30

Page 30 of 43

658

H3, H7 proteins. Binding of FD6 is influenced by adjacent residues DLD, while for

659

HE4 residues QE and IH are important for binding.

660

Figure 6: Antigen-Capture ELISA using FD6 as capture antibody. Recent isolates

662

from HPAIV (H5N1 Th, H5N1 Eg) were well detected, while historic isolates (H5N1

663

Sc and H5N1 Ge) were not (A). The detection of H5N6 by AC-ELISA is concentration

664

dependent (B).

cr

ip t

661

Ac ce pt e

d

M

an

us

665

31

Page 31 of 43

665

Tables

666

Table 1:

667

Influenza viruses used in this study with GenBank protein accession numbers

668

Accession number

human

H5N1

A/Thailand/1(Kan-1)/04

AAS65615

H1N1

A/Fukushima/141/06

ACM17297

A/Brisbane/59/07

ACA28844

A/New Caledonia/20/99 H3N2

A/California/7/04

ip t

Virus strain

cr

Virus subtype

ABH01021

an

A/Wisconsin/67/05

M

A/Brisbane/10/07

ACF54576 ABW23353

A/chicken/Scotland/59

ACZ48553

H5N1

A/teal/Germany/WV632/05

CAL59779

H5N1

A/chicken/Egypt/5610NAMRU-

d

H5N1

Ac ce pt e

Avian

AAP34324

us

Origin

DQ837587

F3/2006

Swine

H5N2

A/duck/Potsdam/1402-6/86

ABI84497

H5N3

A/teal/England/7894/06

Not available

H5N3

A/turkey/Germany/R1612/08

Not available

H5N6

A/duck/Potsdam/2216-4/84

ABB88348

H3N8

A/duck/Ukraine/1/63

ABB88369

H1N1

VL2008/32261-3/1

Not available

H3N2

VL2008/27783-2/4

Not available

669

32

Page 32 of 43

669

Table 2:

670

Sequence of primers used for amplification of truncated HA sequences Primer sequence (5’- 3’)b

P1

P1s

GTA CTC GAG AAG AGA GAG GCT GAA GCA GAT CTA GAT GGA GTG AAG CC

P1as

CAT GCG GCC GCC TTC TCC ACT ATG TAG GAC C

P2s

GTA CTC GAG AAG AGA GAG GCT GAA GCA AAT AAT ACC AAC CAA GAA GAT C

P2as

CAT GCG GCC GCG TCC CCT TTC TTG ACA ATT TTG

P3s

GAA CTC GAG AAG AGA GAG GCT GAA GCA CAC AAT ATA CAC CCT CTC ACC

P3as

CAT GCG GCC GCT CTC TTT TTT CTT CTT CTC TCT C

P4s

GCA CTC GAG AAG AGA GAG GCT GAA GCA AAA GAA TCC ACT CAA AAG GC

P4as

CTT GCG GCC GCT TCC TTT GCA TTA TCC CTA AGC

rHA1s

GTA CTC GAG AAG AGA GAG GCT GAA GCA GAT CAG ATT TGC ATT GGT TAC C

rHA1

cr

us

an

M

P4

d

P3

Ac ce pt e

P2

ip t

PolyPrimer a peptide

rHA1as

GAT GCG GCC GCT CTT TGA GGG CTA TTT CTG AGC C

671

a

672

virus subtype H5N1 (A/Thailand/1(Kan-1)/ 2004).

673

b

674

NotI (antisense primer) used for cloning are written in bold.

P1, P2, P3, P4 and rHA1 DNA fragments coding for HA sequences of influenza A

Gene specific sequences are underlined, restriction sites of XhoI (sense primer) and

675

33

Page 33 of 43

675

Table 3:

676

Designation,

677

haemagglutinin polypeptides P1-P4.

Poly-

location

Location

within

H5

protein

and

sequence

Sequenceb

four

DLDGVKPLIL RDCSVAGWLL GNPMC

us

HA1 165-261

7.59

cr

HA1 43-82

ip t

[kDa]

DEFIN VPEWSYIVEK P2

the

mol wtc

peptidea P1

of

NNTNQEDLLV LWGIHHPNDA AEQTK

16.06

an

LYQNP TTYISVGTST LNQRLVPRIA

TRSKVNGQSG RMEFFWTILK PNDAI

M

NFESN GNFIAPEYAY KIVKKGD HA1 292-330

HNIHPLTIGE CPKYVKSNRL VLATG

d

P3

7.67

P4

Ac ce pt e

LRNSP QRERRRKKR

HA2 38-132

KESTQKAIDG VTNKVNSIID KMNTQ

14.26

FEAVG REFNNLERR IENLNKKMED GFLDVWTYNA ELLVLMENER TLDFH DSNVK NLYDKVRLQL RDNAKE

678

a

679

(A/Thailand/1(Kan-1)/ 2004) haemagglutinin.

680

b

681

using method of Kolaskar and Tongaonkar (1990) are written bold.

P1, P2, P3 and P4 truncated sequences of influenza A subtype H5N1

Epitopes predicted by three of seven algorithms are underlined, epitopes predicted

34

Page 34 of 43

682

c

683

glycosylation site, molecular weight of N-linked oligosaccharide is calculated for the

684

dominant oligosaccharide structure found in Pichia pastoris, i.e. Man9GlcNAc2 with 2

685

kDa (Montesino et al., 1998).

Calculated molecular weight of his-tagged polypeptides. P2 contains a potential N-

ip t

686 687

cr

688

us

689

Table 4:

691

Relative conservation of epitopes of mAbs FD6 and HE4 identified using

692

epitope mapping.

an

690

mAb

Epitopea

94

VKPLI

Ac ce pt e

HE4

H5Nx

H7Nx

H9Nx

H3Nx

H1Nx

49

-

-

-

-

39,9

-

-

-

-

d

H5N1 FD6

M

Relative conservationb [%]

QEDLLVLWGIH

92

693

a

Amino acids important for antibody binding and used for analysis are written bold.

694

b

Relative epitope conservation in isolates from 1990-2013 of subtypes H5N1 (1537

695

isolates), H5Nx (461 isolates, excluding N1-subtype), H7Nx (574 isolates), H9Nx

696

(937 isolates), H3Nx (3673 isolates) including 360 Canadian/American H3N2 variants

697

from swine and H1Nx (5991 isolates) was determined using „Identify Short Peptides

698

in

699

http://www.fludb.org, 16.05.2014, Squires et al., 2007).

Proteins“

tool

provided

by

Influenza

Research

Database

(IRD,

700

35

Page 35 of 43

700

Table 5:

701

Relative sensitivity and specificity between AC-ELISA and RT-PCR

704

Positive

13

0

Negative

3

14

Total

16

14

Relative sensitivity

81%

Relative specificity

100%

cr

13

ip t

Negative

17

M

an

30

d

703

Positive

Ac ce pt e

702

Total

RT-PCR

us

AC-ELISA

36

Page 36 of 43

704

Highlights

705 706

-

Algorithms were used to indentify B-cell epitopes within influenza H5.

707

-

Conserved

708

-

Expressed antigens were used for generation of monoclonal antibodies.

709

-

Two

710

-

Antigen-Capture ELISA for specific detection of H5N1 virus was developed.

expressed

antibodies

specific

for

in

Pichia

H5

pastoris.

were

established.

cr

monoclonal

were

ip t

epitopes

us

711 712

Ac ce pt e

d

M

an

713

37

Page 37 of 43

Ac

ce

pt

ed

M

an

us

cr

i

Figure(s)

Page 38 of 43

Ac

ce

pt

ed

M

an

us

cr

i

Figure(s)

Page 39 of 43

Ac

ce

pt

ed

M

an

us

cr

i

Figure(s)

Page 40 of 43

Ac ce p

te

d

M

an

us

cr

ip t

Figure(s)

Page 41 of 43

Ac

ce

pt

ed

M

an

us

cr

i

Figure(s)

Page 42 of 43

Ac ce p

te

d

M

an

us

cr

ip t

Figure(s)

Page 43 of 43