JVI Accepted Manuscript Posted Online 21 January 2015 J. Virol. doi:10.1128/JVI.03014-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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A novel humanized antibody neutralizes H5N1 via two different

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mechanisms

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Yunrui Tan1, Qingyong Ng1, Qiang Jia1, Jimmy Kwang 1,2*and Fang He1*

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1

Animal Health Biotechnology, Temasek Life Sciences Laboratory, Singapore, Singapore, 2

Department of Microbiology Faculty of Medicine, National University of Singapore, Singapore, Singapore

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Key words: H5N1 AIV; humanized neutralizing antibody; conformational or

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linear epitope

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* Correspondence author: Animal Health Biotechnology, Temasek Life Sciences Laboratory,

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1 Research Link, National University of Singapore, Singapore, 117604.

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Phone: +65-68727470. Fax: +65-68727007. E-mail: [email protected] (Fang He) [email protected] (J.Kwang).

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Abstract

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H5N1 HPAI virus continues to be a severe threat to public health, as well as the poultry

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industry, due to its high mortality and antigenic drift rate. Neutralizing monoclonal antibodies

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can serve as a useful tool in preventing, treating and detecting H5N1. In the present study, a

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humanized H5 antibody 8A8 was developed from a murine H5 monoclonal antibody (Mab).

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Both the humanized and mouse Mabs presented positive activity in HI, virus neutralization

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and IFA against a wide range of H5N1s. Interestingly, both human and murine 8A8 were able

33

to detect H5 in western blotting under reduction conditions. Further, by sequencing escape

34

mutants, the conformational epitope of 8A8 was found to be located within the receptor

35

binding domain (RBD) of H5. The linear epitope of 8A8 was identified by western blotting

36

overlapping fragments and substitution mutants of HA1. RG H5N1s with individual

37

mutations in either the conformational or linear epitope were generated and characterized in a

38

series of assays including HI, post-attachment and cell-cell fusion inhibition assay. The

39

results indicate that for 8A8, virus neutralization mediated by RBD-blocking relies on the

40

conformational epitope while binding to the linear epitope contributes to the neutralization by

41

inhibiting membrane fusion. Taken together, we report in this study that a novel humanized

42

H5 Mab binds to two types of epitopes on HA, leading to virus neutralization via two

43

mechanisms.

44

45

Importance

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Recurrence of the highly pathogenic avian influenza (HPAI) virus subtype H5N1 in humans

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and poultry continues to be a serious concern to public health. Preventive and therapeutic

48

measures against influenza A viruses have received much interest in the context of global

49

efforts to combat the current and future pandemic. Passive immune therapy is considered to

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50

be the most effective and economically prudent preventive strategy against influenza besides

51

vaccination. It is important to develop a humanized neutralizing Mab against all clades of

52

H5N1. For the first time, we report in this study that a novel humanized H5 Mab binds to two

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types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings

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further deepen our understanding of influenza neutralization.

55

Introduction

56

Recurrence of the highly pathogenic avian influenza (HPAI) virus subtype H5N1 in humans

57

and poultry continues to be a serious concern to public health due to its unabated and

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widespread geographical circulation (9). Since their emergence in Asia over a decade ago,

59

highly pathogenic avian influenza H5N1 viruses have spread to over sixty countries on three

60

continents and are endemic among poultry in South East Asia and Africa (34). They have

61

caused disease in several mammals, including humans, often with lethal consequences. Up to

62

date, H5N1 has resulted in 667 human cases worldwide, including 393 deaths (42). Although

63

so far no sustained human-to-human transmission of the virus has been observed, the concern

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remains that, if human transmissibility was acquired, a severe pandemic could occur (16, 25).

65

Preventive and therapeutic measures against influenza A viruses have received much interest

66

in the context of global efforts to combat the current pandemic and to prevent such a situation

67

in the future. Given the emerging occurrence of oseltamivir/zanamivir-resistant viruses (30)

68

and the high and long-term dosing requirements for antiviral drugs (45), vaccination and

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passive immune therapy are considered to be the most effective and economically prudent

70

preventive strategy against influenza (3). However, vaccine strategies are usually hindered by

71

antigenic variation (36) and cannot deliver immediate efficacy against acute infection caused

72

by several influenza subtypes. These subtypes include H5N1 and H7N9 (20), which develop

73

severe disease quickly within days of infection. The use of monoclonal antibodies in the

3

74

treatment of medical condition has been well established for viral infectious diseases,

75

including HIV (44) and hepatitis (7). Therefore, administration of monoclonal antibodies

76

against neutralizing epitopes may be an attractive alternative for influenza treatment (32),

77

especially in the case of individuals who are at high risk from influenza infections. Such

78

individuals include immuno-compromised patients or the elderly who do not generally

79

respond well to active immunization (29).

80

Influenza hemagglutinin (HA), the principal determinant of immunity to the influenza virus,

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is the main target and antigenic source for neutralizing antibodies against viral infections (15).

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HA is generated as a single polypeptide and folds into a trimeric spike (HA0) which is

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subsequently cleaved into HA1 and HA2 subunits by host proteases during infection. The

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globular head domain of HA molecule is composed of HA1 subunits and is the most

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immunogenic part of HA. This globular head contains the receptor binding domain, which

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mediates viral attachment to the host’s cell sialic-acid receptors (12). Antibodies binding to

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these regions are usually strain-specific and very few cases show broad neutralization activity,

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even within a single subtype. HA2 and several HA1 residues form a mostly helical stem

89

region, which supports the core fusion machinery. Most stem binding antibodies present a

90

remarkably broad neutralizing activity against influenza viruses of different subtypes (28).

91

Mabs targeting these regions are usually able to neutralize the influenza virus by physically

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interrupting either receptor binding or membrane fusion, two key functions of HA (40).

93

Humanization and human antibody production are crucial procedures in passive immune

94

therapy since murine antibodies fail to trigger a proper human immune response and instead

95

elicit a human anti-mouse antibody response (24, 31). However, one current challenge is that

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humanization often leads to loss or changes in effector activity of Mabs. Preserving the

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correct functional CDRs of the original murine antibodies while minimizing the murine

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immunogenic elements in antibodies are critical goals in the production of human therapeutic

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antibodies (43).

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In the present study, a human H5 Mab 8A8 was generated from a neutralizing murine Mab by

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CDR grafting and humanized replacement in the V regions. Both human and murine Mabs

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presented broad neutralizing activity against different clades of H5N1s and showed positive

103

reactivity to reduced H5 in western blots. Using epitope mapping by either escape mutants or

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HA fragments, different conformational and linear epitopes for the same antibody 8A8 were

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identified. Neutralization mechanisms related to each type of epitope were further studied, as

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discussed below.

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Materials and Methods

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Production and characterization of murine Mab

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MAb was produced as described previously. Briefly, BALB/c mice were immunized twice

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with two weeks interval by the subcutaneous injection of individual BEI-inactivated H5N1

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virus (A/Indonesia/CDC669/2006) mixed with Montanide ISA563 adjuvant (Seppic, France).

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Mice received an additional intravenous injection of the same viral antigen 3 days before the

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fusion of splenocytes with SP2/0 cells. Hybridoma culture supernatants were screened by

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immunofluorescence assays. Hybridomas that produced specific MAbs were cloned by

116

limiting dilution, expanded, and further subcultured. The hybridoma culture supernatant was

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clarified and tested for the hemagglutination inhibition activity as described below.

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Production and characterization of human Mab

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The human Mab 8A8 was generated by service collaboration with Antitope, Ltd., UK based

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on a murine H5 Mab. Briefly, RNA was extracted from murine 8A8 hybridoma cell by using 5

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RNAqueous® -4PCR kit (Ambion cat. no. AM1914). mRNA of the IgG heavy chain variable

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region was amplified using a set of six degenerated primer pools (HA to HF) while the IgkVL

123

light chain V-region was amplified using a seven degenerate primer pools (KA to KG). PCR

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products were sequenced by pGEM-T® Easy vector (Promega cat no. A1360). A single

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functional heavy chain variable region (VH) gene sequence and a single functional kappa

126

light chain variable region (Vk) gene sequence were identified from the sequencing analysis.

127

Structural models of the mouse 8A8 antibody V-regions were produced using Swiss PDB and

128

the "constraining " amino acid residues in V region that were likely to be essential in the

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antibody-antigen binding properties were identified. Based on the analysis, only a few

130

corresponding sequence segments from human antibodies sequence were identified as

131

possible alternative residues within Complementarity-Determining Regions (CDRs). VH and

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Vk sequence segments of 8A8 Composite Human antibody variants were selected from

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Antitope's database of unrelated fully humanized antibodies and analyzed using iTope™

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technology in silico (Perrt et al 2008). Sequences that were categorized as significant non-

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human germline binders to human MHC class II alleles were discarded. In silico analysis by

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using TCED™ (T Cell Epitope Database) of known antibody sequence-related T cell

137

epitopes was carried out to eliminate those variants which contain potential T cell epitopes

138

within the sequence segments and also within the junctions between the segments. Five VH

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sequence variants and four Vk sequence variants were selected for humanized Ab expression.

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DNA fragments encoding humanized variant VH and Vk regions were synthesized and were

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cloned into IgG1 expression vectors pANT17.2 and pANT13.2 respectively. The VH region

142

was cloned using MluI and HindIII restriction sites, and the Vk region was cloned using

143

BssHI and BamHI. Recombinant constructs of heavy chain and light chain were stably co-

144

transfected into NS0 cells via electroporation and selected using 200nM methotrexate (Sigma

6

145

cat no. M8407). Expressed humanized Ab variants were screened in HI for the candidate with

146

highest yield and activity.

147

Viruses and cell lines

148

H5N1 human influenza viruses A/Indonesia/CDC669/2006 and other viruses of clade 2.1

149

were obtained from the Ministry of Health (MOH), Republic of Indonesia. The H5N1 viruses

150

from different phylogenetic clades or subclades were rescued by reverse genetics. Briefly, the

151

hemagglutinin (HA) and neuraminidase (NA) genes of H5N1 viruses from individual clades

152

were synthesized (GenScript) based on the sequences in the NCBI influenza virus database.

153

The synthetic HA and NA genes were cloned into a dualpromoter plasmid for influenza A

154

virus reverse genetics (38). The dual-promoter plasmids were obtained from the Centers for

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Disease Control and Prevention, Atlanta, GA. Reassortant viruses were rescued by

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transfecting plasmids containing HA and NA along with the remaining six influenza virus

157

genes derived from high-growth master strain A/Puerto Rico/8/34 (H1N1) into cocultured

158

293T and MDCK cells by using Lipofectamine 2000 (Invitrogen Corp.). At 72 h

159

posttransfection the culture medium was inoculated into embryonated eggs or MDCK cells.

160

The HA and NA genes of reassortant viruses from the second passage were sequenced to

161

confirm the presence of the introduced HA and NA genes and the absence of mutations.

162

Stock viruses were propagated in the allantoic cavity of 10-day-old embryonated eggs, and

163

virus containing allantoic fluid was harvested and stored in aliquots at 80°C. Virus content

164

was determined by a standard hemagglutination assay as described previously (19).

165

MDCK cells were maintained in Dulbeccos Modified Eagle Medium (DMEM; Life

166

Technologies, USA) containing 10% Fetal Bovine Serum (FBS; Life Technologies, USA).

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293T were maintained in Opti-MEMI (Life Technologies, USA) containing 5% FBS. After

168

48 h the transfected supernatants were collected and virus titers were determined by standard

7

169

hemagglutination assays. The tissue culture infectious dose 50 (TCID50) of reassortant virus

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was then calculated by the Muench-Reed method (1938).

171

Hemagglutination inhibition assay (HI)

172

Hemagglutination inhibition assays were performed as described previously (39). Briefly,

173

Mabs or receptor-destroying enzyme (RDE)-treated sera were serially diluted (2 fold) in V-

174

bottom 96-well plates and mixed with 4 HA units of H5 virus. Plates were incubated for 30

175

min at room temperature, and 1% chicken RBCs were added to each well. The

176

hemagglutination inhibition endpoint was the highest antibody dilution in which

177

agglutination was not observed.

178

Microneutralization assay

179

Neutralization activity of Mab or serum against H5 strains was analyzed by

180

microneutralization assay as previously described (21). Briefly, antibody samples were

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serially two-fold diluted and incubated with 100 TCID50 of different clades of H5 strains for

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1 h at room temperature and plated in duplicate onto MDCK cells grown in a 96-well plate.

183

The neutralizing titer was assessed as the highest antibody dilution in which no cytopathic

184

effect was observed by light microscopy.

185

Immunofluorescence assay (IFA)

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MDCK cells cultured in 96-well plates were infected with AIV H5 strains. At 24 h post-

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infection, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and

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washed thrice with phosphate buffered saline (PBS), pH 7.4. Fixed cells were incubated with

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hybridoma culture supernatant at 37 ºC for1 h, rinsed with PBS and then incubated with a

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1:200 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse or anti-

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human Immunoglobulin (Dako, Denmark). Cells were rinsed again in PBS and antibody

192

binding was evaluated by wide-field epi-fluorescence microscopy (Olympus IX71).

193

SDS PAGE

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SDS-PAGE was performed as described previously (2)using a discontinuous buffer system

195

with a 12% polyacrylamide separating gel. The protein samples were prepared by mixing

196

with 2X SDS sample buffer (0.2 M Tris-HCl PH 6.8, 8% SDS, 40% glycerol, 0.6 M β-

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mercapthanol, 0.05 EDTA, 0.04% bromophenol blue) and heating at 100°C for 10 min.

198

Western blotting

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Cell lysates of IPTG-induced E. coli cultures or H5N1 viruses were separated by 12% sodium

200

dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins in the gel were then

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transferred onto a nitrocellulose membrane and blocked with 5% nonfat milk in PBST (1X

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PBS and 0.1% Tween 20) for 1 h at room temperature. The membrane was incubated with

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MAb supernatant, rinsed with PBST, and incubated with horseradish peroxidase-conjugated

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rabbit anti-mouse or anti-human immunoglobulin G (IgG) (Dako, Denmark) for 1 h at room

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temperature. Following washing with PBST, the membrane was developed by incubation

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with ECL reagents (Amersham Biosciences).

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Immunodot Blotting

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Protein samples were loaded onto the 0.45 mm nitrocellulose membrane (Bio-rad) using a 96-

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well hybridot manifold (BIORAD). The membrane was then blocked with 5% nonfat milk in

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PBS containing 0.1% tween 20 (PBST) at room temperature for 30 min. Rinsing was done

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with PBST for 3 times. The membrane was further incubated with monoclonal antibodies in

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PBST at room temperature for 1 hr. Following rinsing, the membrane was incubated with

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213

corresponding secondary antibody conjugated with HRP for 1 hr at room temperature. Bound

214

antibodies were detected by incubation with ECL reagents (Amersham Biosciences).

215

Isolation and analysis of escape mutants

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The conformational epitope recognized by Mab 8A8 was mapped by characterization of

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escape mutants as described previously He et al. (2010). Briefly, H5N1 parental virus

218

(A/Indonesia/CDC669/06) was incubated with an excess of Mab for 1 h and then inoculated

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into 11 days old embryonated chicken eggs. The eggs were incubated at 37 ºC for 48 h. Virus

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was harvested and used for cloning in limiting dilution in embryonated chicken eggs and the

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escape mutants were plaque purified. The HA gene mutations were then identified by

222

sequencing and comparison with the sequence of the parental virus.

223

Linear epitope mapping

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Overlapping

225

A/Indonesia/CDC669/06 (H5N1) (Fig. 2A) were amplified by PCR and cloned into the pET-

226

28a (+) vector. The peptides were expressed in E. coli and analyzed by Western blotting. A

227

panel of mutants was generated by substitution of amino acids in test positions with site-

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directed mutagenesis using a commercial kit (QuikChange; Stratagene), and the introduced

229

mutations were confirmed by sequence analysis. The mutants generated were further tested in

230

Western blotting.

231

Postattachment assay

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50ul of virus suspension containing 1000 TCID50 of H5N1 was added to MDCK cells in one

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well of 96-well plate, followed by incubation at 4°C for 1 h to allow virus attachment to cells.

234

The plate was carefully washed for three times. Subsequently, 50 ul of serially diluted Mabs

235

were added to the wells and incubated at 37°C for 1 h. After rinsing three times, 100ul of

fragments

encoding

the

open

reading

frame

of

the

HA

of

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DMEM plus 2% FBS was added to the plate/well, followed by incubation at 37°C with 5%

237

CO2. After 24 h, the infection and inhibition effects were observed and determined by IFA.

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Protease susceptibility assay

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The protease susceptibility assay was modified from the method described by Edwards and

240

Dimmock (11). Briefly, H5N1 strains were incubated with antibody at 37°C for 1 h. The pH

241

of the neutralization mixture was then either adjusted to pH 8 or lowered to pH 5 by the

242

addition of 1 M HCl at 37°C for 45 min. The pH was then recovered to neutrality. The low-

243

pH induced conformational change of HA was detected by incubation with proteinase K

244

(0.5ug/ml, NEB) at 37°C for 30 min. The digestion was stopped by the addition of SDS

245

loading dye and was further analyzed in Western blotting.

246

Cell-cell Fusion Inhibition assay

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A cell fusion inhibition assay was performed as described previously (8). In brief, Vero cells

248

were grown to 80% confluent in 24-well plates and transfected with pcDNA-H5 plasmid (1.6

249

mg total DNA per well) using lipofectamine 2000 (Invitrogen) according to the instruction

250

manual. Transfected cells were maintained in medium supplemented with 0.5 mg/ml G418

251

(iDNA). After 48 hours of transfection, the culture medium was supplemented with serially

252

diluted h8A8 or a control Mab for 1 h. After washing three times by PBS (pH 7.4), cells were

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incubated with low-pH fusion inducing buffer (150 mM NaCl, 10 mM HEPES, adjusted to

254

pH 5.0) for 5 mins, and returned to the standard culture medium for 2 hours at 37ºC. Finally,

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cells were fixed with 4% polyoxymethylene and stained with 0.5% crystal violet for 20 mins.

256 257

Results

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Humanized Mab 8A8 sustains the same activity to H5N1 as murine 8A8

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The murine Mab 8A8 (m8A8), which belongs to IgG1, revealed a pattern of cytoplasmic

260

staining by IFA in MDCK cells infected with A/Indonesia/CDC669/06 (H5N1, CDC669). H5

261

specificity was verified by IFA with non-H5N1 influenza infected MDCK cells. The m8A8

262

antibody demonstrated positive activity in virus neutralization, HI (Table1 and 2) and western

263

blotting against the CDC669 whole virus (Fig. 1A and B). The recognition spectrum was

264

further evaluated in HI with a wide range of H5N1 of different clades. As shown in Table 1,

265

m8A8 was able to react to all H5N1s tested from clade 1.0 to 9 without cross-reactivity with

266

any non-H5N1 strains as evaluated by HI. Based on all these significant features and in order

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to generate an effective reagent for human therapy, m8A8 was converted into a human IgG1

268

Mab 8A8 (h8A8) with minimal murine immunogenicity by CDR grafting and V region

269

replacement. Secreted h8A8 was tested in IFA, HI, virus neutralization and western blotting

270

for activity verification. h8A8 successfully detected H5 expression in CDC669-infected

271

MDCK cells with no reactivity to non-H5N1 strains in IFA. Equal concentrations of purified

272

h8A8 and m8A8 were both able to neutralize H5N1s at a comparable titer in both HI and

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virus neutralization assays (Table 2), suggesting the involvement of a conformational epitope

274

(37). However, interestingly, both m8A8 and h8A8 were able to recognize either recombinant

275

H5 HA1 or H5 whole virus in a Western blot, suggesting that 8A8 targets a linear epitope on

276

H5.

277

A dot blot with H5 in different conditions was performed to determine the reactivity of h8A8

278

to native and reduced H5. As shown in Fig.1C, h8A8 could strongly detect denatured E. coli-

279

expressed H5 in both highly and lowly reducing buffers after heating, but the reactivity to

280

untreated E.coli-expressed H5 was weak. In contrast, when probed with h8A8, strong dots

281

were observed with native baculovirus-expressed H5 (Bac-H5) which possesses a

282

conformation similar to H5 from native H5N1 (22). However, the intensity of dots with

283

reduced Bac-H5 decreased significantly when the same blot was probed with h8A8. These 12

284

observations indicate that h8A8 is able to bind either H5 in its native conformation or

285

reduced H5 via the exposed linear epitope.

286

Overall, these results indicate that humanized 8A8 preserves the binding properties of m8A8

287

to H5N1 as confirmed by different tests. Also, 8A8 is confirmed to be capable of binding H5

288

in both native and reduced conditions.

289

Identification of both conformational and linear epitopes for h8A8

290

To discover the epitope and related binding sites on H5 for 8A8, epitope mapping was

291

performed with two different methods. The conformational epitope was identified using

292

neutralization escape mutants. Taking CDC669 as the parental strain, escape mutants were

293

generated from the selection with h8A8. Evasion was confirmed in HI with h8A8 (Table 4).

294

The complete HA genes of the cloned escape mutants were sequenced. HA amino acid

295

numbering in this work uses H5 numbering excluding the signal peptide. Mutant clones

296

showed three types of mutations: double mutant at Arg189 and Ser223, single mutant at

297

Lys218 and single mutant at Ser223 (Table 3). This indicated that the three amino acids are

298

involved in forming the conformational epitope of 8A8.

299

Meanwhile, the linear epitope was mapped with a set of overlapping open reading frame

300

expression clones and single mutants for recombinant H5 HA1. Fragments of H5 were

301

designed and probed with h8A8 as shown in Fig.2. h8A8 reacted to F1 but not to any of the

302

rest (F2-F5), indicating that the epitope comprised amino acids from aa250 to aa290. Sub-

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fragments A to H were further generated to refine the epitope and revealed that h8A8 targets

304

amino acids upstream of aa258. A panel of HA1s with single substitutions from aa243 to

305

aa260 were subsequently constructed, expressed and analyzed by Western blotting with h8A8.

306

The results showed that h8A8 failed to react with individual HA1 carrying single amino acid

307

substitutions from aa244 to aa256, with the exception of the E251A mutant. Two mutants at 13

308

the same position, E251R and E251W, were further tested to be positive in Western blotting

309

with h8A8, suggesting the contribution of aa251 in H5 recognition by h8A8 is minor. Taken

310

together, these findings indicate that the linear epitope of h8A8 ranges from aa244 to aa256.

311

The two types of h8A8 epitopes are independent of each other

312

Vero cells were transfected with constructs expressing H5s with individual mutated epitope.

313

HA expression was probed with either h8A8 or control H5 antibodies targeting the N-

314

terminal of H5. As shown by IFA (Fig. 3A), h8A8 detected H5 expression in Vero cells

315

transfected individually with these epitope mutants with the exception of the double mutants

316

in both types of epitopes. In IFA, both native and denatured HA are presented together in

317

cells. Hence, this result indicates that any disruption in one type of epitope does not largely

318

affect h8A8 recognizing the other type. Binding is only abolished when both epitopes are

319

mutated.

320

To confirm the independent binding of the two epitopes, a peptide fragment expressing the

321

linear epitope only was designed and probed with h8A8 as shown in Fig. 3B. In Western blot,

322

h8A8 successfully detected the fragment (aa230-330) in which the conformational epitope

323

was completely excluded. With the same protein quantity, the signal density of the fragment

324

band was similar to the full-length HA1 in the blot probed with h8A8. Therefore, the result

325

confirmed that the interaction to the linear epitope by h8A8 is independent of the

326

conformational epitope.

327

The 3D structure of CDC669 H5 was generated by Swiss Model. Amino acid positions

328

involved in forming either the conformational or linear epitope were highlighted in the HA

329

structure (Fig. 4A and 4B). Aa218 and aa223 from the conformational epitope are both

330

located in 220-loop. Together with aa189, the three amino acids are found within Receptor

331

Binding domain (RBD) of H5 (41). Further, aa189 is located in antigenic site Sb (23). In 14

332

contrast, none of amino acids from the linear epitope are located in RBD or any antigenic site.

333

The whole linear epitope is not exposed in the structure of mature HA. As shown in Fig. 4A

334

and 4B, the central part of the linear epitope was internalized behind other amino acids. This

335

observation suggests that the interactions by h8A8 to the two types of epitopes do not occur

336

simultaneously. Therefore, the results further confirm that the conformational and linear

337

epitopes of h8A8 are independent of each other.

338

To study the conservation of the epitopes, an alignment was performed for H5 protein

339

sequences of different clades (Fig. 4C). Aa218 and aa223, the two single mutations causing

340

virus evasion, were found to be conserved among different major clades, while position

341

aa189 from the double escape mutant tended to be variable. The 13 amino acid linear epitope

342

was generally conserved among these clades with sporadic variations at two positions, aa 249

343

and aa 252.

344

The conformational epitope of 8A8 is responsible for receptor binding

345

Because the amino acids of the conformational epitope are found within antigenic sites and

346

the RBD domain, it is believed that the antibody binding the conformational epitope should

347

inhibit virus attachment to host cells. To further characterize the identified epitopes, a panel

348

of RG influenza viruses based on CDC669 H5 was generated with individual mutations

349

covering various amino acids from the two epitopes. Hemagglutination inhibition (HI) assays

350

and virus neutralization were thus performed with h8A8 and the panel of RG mutants for the

351

identified epitopes (Table 4). The h8A8 antibody failed to inhibit hemagglutination of three

352

RG mutants, which were generated according to escape mutants (R189KS223R, K218Q and

353

S223R) but successfully stopped hemagglutination of mutants for the linear epitope (N244A

354

and I256A). Similarly, in virus neutralization assays, h8A8 neutralized the mutants for the

355

linear epitope but not the RG escape mutant K218Q. Interestingly however, h8A8 could fully

15

356

neutralize the mutants S223R and R189KS223R which are not reactive to h8A8 in HI,

357

suggesting other neutralization machinery is employed. Taken together, the results confirm

358

that the conformational epitope identified for h8A8 is responsible for receptor binding and

359

that h8A8 blocks the RBD by binding to the conformational epitope, leading to virus

360

neutralization. Also, the results imply that h8A8 uses a neutralization mechanism which does

361

not depend on RBD blocking and is independent of the conformational epitope.

362

The linear epitope of 8A8 is involved in member fusion

363

In order to identify other neutralization mechanisms for h8A8, a post-attachment assay was

364

first performed with h8A8 against CDC669 and the panel of RG mutants for the epitopes (Fig.

365

5). With the incubation of h8A8 in MDCK cells after virus attachment, infection of CDC669

366

was completely inhibited at a dose of 10ug and partially inhibited with 1ug of h8A8. Mutant

367

K218Q escaped from this post-attachment neutralization, consistent with its escape from

368

neutralization discussed earlier. S223R, the other conformational epitope mutant, showed

369

similar inhibition as wild type CDC669, indicating that aa223 from the conformation epitope

370

is not involved in post-attachment neutralization. In contrast, the high dose of h8A8 failed to

371

abolish the infection of the mutant in the linear epitope mutant (N244A) and only a slight

372

decrease in infection was observed with the low dose of h8A8, indicating the linear epitope

373

contributes to post-attachment neutralization by h8A8.

374

Because inhibition in membrane fusion is the major mechanism of post-attachment

375

neutralization, protease susceptibility and cell-cell fusion inhibition assays were further

376

performed. In the protease susceptibility assay, the H5 protein was exposed to a low pH (pH

377

5.0) to trigger pH-induced conformational changes and susceptibility to degradation by

378

protease K. As shown in Fig.6A, when incubated with 3H12, a control antibody with HI

379

activity, CDC669 H5 was detected in the sample at pH 8.0 but not at pH 5.0. In contrast,

16

380

upon pre-incubation with h8A8 before the exposure to pH5.0, the degradation of CDC669 H5

381

was inhibited. Before the protease assay, CDC669 and RG epitope mutants were first tested

382

in western blotting with h8A8 (Fig.6B). h8A8 is able to detect mutants in the conformational

383

epitope as well as the wild type CDC669. In contrast, no band was observed in any of the

384

linear epitope mutants probed by h8A8. h8A8 was further incubated with a range of epitope

385

mutants for the degradation susceptibility study (Fig.6C). Each mutant was tested at the same

386

HA titer of 26. Significant degradation of H5 upon low pH treatment was observed in N244A

387

and I256A, the two mutants for the linear epitope, whereas conformational epitope mutants

388

treated with low pH were detected at a similar density compared to the groups treated at pH

389

8.0. These observations indicate that h8A8 binding is able to protect H5 from degradation at

390

low pH and such protection is mediated by interaction with the linear epitope. This is the key

391

indicator of membrane fusion inhibition (13).

392

In the cell-cell fusion inhibition assay (Fig. 7), h8A8 of both high and low doses successfully

393

inhibited the cell-cell fusion caused by H5 conformation change upon low pH, while the

394

control antibody 3H12 failed to stop the fusion. The complete escape mutant K218Q did not

395

shown any interference in the fusion by either h8A8 or 3H12. Fusion by the conformational

396

epitope mutant S223R was significantly inhibited by h8A8 but not by 3H12. H8A8 partially

397

reduced the fusion caused by N244A H5, the mutant in the linear epitope, even at the high

398

dose. Taken together, these results confirm that h8A8 is able to disturb HA-mediated

399

membrane fusion and also that the linear epitope is involved in the inhibition.

400

401

Discussion

402

As is the case with the majority of neutralizing antibodies, hemagglutinin serves as the most

403

important target in passive immune therapy as well as in vaccine development against HPAI 17

404

viruses, such as H5N1 (14). RBD blocking by head-binding Mabs is one of the most

405

employed mechanisms by antibodies for influenza virus neutralization. Three other

406

mechanisms have been characterized for hemagglutinin targeted neutralization, including

407

inhibition in membrane fusion, HA cleavage and virus egress (4). In this study, a unique Mab

408

was found to react to two types of epitopes on HA and to neutralize H5N1 via two different

409

mechanisms. This humanized antibody presents the same immunological activity and

410

neutralizing features as its original murine antibody, providing a useful tool for effective and

411

efficient antibody therapy against H5N1.

412

Based on the results, it is believed that the functions of two epitopes targeted by 8A8 are

413

independent of each other, instead of being synergistic in neutralization. Firstly, the two

414

epitopes do not exist simultaneously in one HA subunit. The conformational epitope appears

415

only in the native HA structure at the time when the linear epitope is shielded by other amino

416

acids on the surface. The linear epitope is exposed when HA conformation changes by either

417

natural processes or environmental factors. Certain biological processes leading to changes in

418

HA structure include membrane fusion by low pH and potential virus “breath”. Studies in

419

enveloped virus suggest that mature virions are dynamically “breathing” (10), a reference to

420

regular motions among amino acids in enveloped proteins. Since the influenza virus is

421

enveloped, similar dynamics could occur in glycoproteins presented on the viral surface.

422

When the exposed linear epitope is bound by Mabs, the related biological process will be

423

disrupted, leading to virus neutralization. Environmental factors, such as heat or chemical

424

contact, will also denature HA and expose the linear epitope (18). Therefore, 8A8 is able to

425

efficiently recognize H5 from samples in different conditions, making it a useful detection

426

antibody. Further, mutations in the linear epitope do not affect RBD mediated neutralization

427

as indicated in HI, confirming that the linear epitope plays no role in head blocking by 8A8.

428

Similarly, mutations in the conformational epitope do not abolish 8A8 reactivity to HA in 18

429

western blotting, showing that the interaction to denatured HA does not rely on the

430

conformational epitope at all. Taken together, it is concluded that 8A8 binds to two different

431

types of epitopes respectively in different conditions.

432

The two types of epitopes which act individually lead to the same virus neutralization

433

efficacy, but via two separate mechanisms. As identified in escape mutants and confirmed in

434

HI evasion, the conformational epitope is linked to RBD-mediated neutralization. Further, the

435

H5 structure studies indicate that the amino acids identified by escape mutants are located

436

within the RBD domain. This confirms that 8A8 binding blocks RBD and inhibits virus

437

attachment to cells, leading to virus neutralization. Escape mutants R189KS223R and S223R

438

were initially selected based on HI evasion from 8A8. Interestingly, h8A8 failed to react with

439

mutants R189KS223R and S223R in the HI test but succeeded in neutralizing both in MDCK

440

cells, suggesting that other machinery is involved in 8A8-induced virus neutralization. One

441

way in which the influenza virus is neutralized could be through interference with the low-

442

pH-induced conformational change in the HA molecule, resulting in the inhibition of fusion

443

during viral replication (6). A few neutralizing antibodies have been reported to employ this

444

strategy, most of which target epitopes either in the C terminal of HA1 or N terminal of HA2.

445

As shown in the previous study by Tan’s group (33), Mab 9F4 was able to neutralize H5 by

446

inhibiting membrane fusion. 9F4 recognizes the epitope “IVKK” from 256aa to 259aa. The

447

epitope is next to the linear epitope identified here with one amino acid overlapping,

448

implying that the linear epitope of 8A8 is involved in the same mechanism. The protease

449

susceptibility assay demonstrated that 8A8 could prevent HA degradation caused at low pH

450

and that such function relied on the linear epitope being intact. The cell-cell fusion inhibition

451

assay corroborates the theory that h8A8 inhibits membrane fusion and it indicates that the

452

linear epitope mutant is less responsive to inhibition than wild type CDC669. However, no

453

reduction was observed with h8A8 in neutralization titer against mutants of the linear epitope, 19

454

suggesting the role of the linear epitope in total neutralization is minor, as compared to RBD-

455

mediated neutralization. Therefore, it is concluded that 8A8 interaction with the

456

conformational epitope is responsible for blocking RBD and that binding of the linear epitope

457

leads to the inhibition of membrane fusion.

458

8A8 has broad neutralization activity and recognition of H5 from different clades, which

459

relies on these conserved epitopes. Amino acids in RBD are usually not conserved due to

460

antigenic drift (27). However, the two amino acids identified in escape single mutants, 218K

461

and 223S, are highly stable among different clades of H5N1s even though they are within

462

RBD. This may be caused by the possible deficiency in infectivity or replication of these

463

escape mutants. As noticed in IFA with infected MDCK cells (data not shown), weaker H5

464

expression was detected in S223R infection by both 8A8 and control H5 Mab as compared to

465

wild type CDC669, suggesting that the mutation in 223R may not be selected for among

466

H5N1s. Because the conformational and linear epitopes are independent, the linear epitope

467

serves as an additional binding site for 8A8, thus extending the recognition spectrum of 8A8.

468

The linear epitope is conserved among most clades with sporadic variations in only two

469

amino acids. Some mutations are compatible to 8A8 binding, such as those on 251E.

470

Therefore, based on the two types of conserved epitopes, 8A8 is able to neutralize and

471

recognize a wide range of H5N1s from all the major clades.

472

In order to minimize murine immunogenicity in monoclonal antibody, humanization is a

473

required step for passive immune therapy. However, the original activity of murine antibodies,

474

including the neutralizing efficacy, may be altered during the humanization steps. In this

475

study, CDR graft and substitution of murine T cell epitopes (5) were performed to Mab 8A8

476

for complete humanization. Reactivity of h8A8 to H5N1 is comparable to m8A8 as evaluated

477

by IFA, WB, HI and virus neutralization. Hence, humanization based on a fine substitution of

478

murine antigenic residues using TCED™ (35) was found to be successful without any 20

479

disturbance in H5 binding affinity. Therefore, h8A8 can serve as a safe and effective

480

therapeutic agent for human treatment against lethal H5N1 infection.

481

In summary, it was reported in the study that h8A8 is a fully humanized antibody with strong

482

neutralization activity and broad cross-clade protection against H5N1. These advantages of

483

h8A8 come from the dual recognition of two different types of epitopes, which cause virus

484

neutralization via two different mechanisms. The is the first report to our knowledge showing

485

that a H5 antibody is able to bind to two types of epitopes for neutralization using different

486

mechanisms and increases our understanding of HA-mediated neutralization among influenza

487

viruses. Besides passive therapeutics and prophylactics, 8A8 can also be applied to sensitive

488

and specific detection of H5N1 from various clades. The linear and conformational epitopes

489

allow 8A8 to detect H5N1 in different forms and conditions, including native and denatured

490

samples. In the next study, efforts will be made to further characterize the antiviral and

491

diagnostic functions of 8A8 at the pre-clinical level.

492

493

Competing Interests

494

Authors claim no conflict interests.

495 496

Authors’ contributions

497

Conceived and designed the experiments: FH, JK. Performed the experiments: YRT, QYN,

498

FH and QJ. Analyzed the data: FH, JK. Wrote the paper: FH.

499

21

500

Acknowledgements

501

This work was supported by Temasek Life Sciences laboratory, Singapore. We greatly thank

502

Profs. J Sivaraman and K Swaminathan from National University of Singapore and Dr.

503

Xiaowei Li from Xiamen University, China for their advices. We greatly thank Dr. Ian

504

Cheong from Temasek Life Sciences laboratory, Singapore for his proofreading for the

505

manuscript.

506

507

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643 27

644

Figure Legends

645

Figure

646

Immunofluorescence assays in MDCK cells infected with H5N1 (CDC669) or H7N7. Cells

647

were fixed 24h post-infection and incubated with each primary antibody as listed, followed

648

with FITC secondary antibody staining. (B) Western Blotting with m8A8 and h8A8 against

649

H5N1 (CDC669) or H7N7. Each lane was loaded with 20ul of viruses at HA titer of 25. (C)

650

Immunodot blotting with h8A8 against native H5, H5 in lowly reduced buffer (LRB, 2% SDS)

651

and highly reduced buffer (HRB, 0.2 M Tris-HCl PH 6.8, 8% SDS, 40% glycerol, 0.6 M β-

652

mercapthanol, 0.05 EDTA). 2ug or 10ug of E.coli expressed H5 was loaded in one dot. 16

653

HAU or 64 HAU of baculovirus expressed H5 (Bac-H5) was loaded in one dot. RT: room

654

temperature; 100ºC: Heated at 100ºC for 10 minutes.

655

Figure 2. Epitope mapping for h8A8. (A) Plan for epitope mapping and western blotting

656

results. The gene segments coding for fragments 1 to 5 and sub-fragments A-H were

657

expressed as histidine tagged proteins. The cell lysates of bacteria expressing the fragments

658

were tested in Western blotting with h8A8 to map the epitope (the numbers indicate the

659

amino acid number of HA1 without the signal peptide). (B) Western blotting with mutated

660

HA1. Single alanine substitution was made individually in HA1 from 243 aa to 260 aa.

661

Mutated HA1s were expressed as histidine tagged proteins. Numbers listed in each lane

662

indicated each mutant with mutated amino acid position accordingly. HA1: His-tagged HA1

663

from wild type CDC669.

664

Figure 3. Independent binding to the two types of epitopes by h8A8. (A) Reactivity of h8A8

665

to a range of epitope mutants in IFA. Immunofluorescence assays in Vero cells transfected

666

with epitope mutants were performed using h8A8 and other H5 antibodies. 7H10 and 3H12

667

were mixed together as positive controls. (B) Western blot with h8A8 against HA1 peptide

1.

h8A8

preserves

similar

binding

activity

to

H5N1

as

m8A8.

(A)

28

668

fragment excluding the conformational epitope (aa230-330). HA1: full length HA1 of

669

CDC669. UID: the sample before induction as the negative control.

670

Figure 4. Epitope location and sequence analysis. (A) Side view and (B) Top view of

671

CDC669 H5 structure. HA1 (1~330aa) was colored in pink and HA2 was colored in lemon.

672

The other two monomers are shown in white. Receptor binding domain is colored in yellow.

673

The conformational epitope (218aa, 223aa and 189aa) is in red while the linear epitope

674

(244~256aa) in marine. The conformational epitope is overlapped with RBD. The structure

675

was generated by Swiss Model (http://beta.swissmodel.expasy.org) (1, 17, 26), The diagram

676

were generated by PyMOL program (http://www.pymol.org/). (C) Identification of epitopes

677

in antigenic sites among different clades. H5 protein sequences of different major clades were

678

aligned. Amino acid consensus sequences of H5N1 HA clades were highlighted at positions

679

equivalent to the H1 antigenic sites, Ca (in blue boxes) and Sb (in green boxes). The linear

680

epitopes is highlighted in red box and the conformational epitope related amino acids are

681

shown in yellow boxes.

682

Figure 5. Postattachment neutralization assays with h8A8 in MDCK cells. 3H12: a HI

683

antibody against H5N1 used as control. Wild type CDC669 and RG H5N1s with individual

684

mutations in either the conformational or linear epitope were tested. Infection was visualized

685

with immunofluorescence staining using Mab 7H10.

686

Figure 6. Protease susceptibility assays with H5 and h8A8. H5 was visualized in Western

687

blotting with Mab 7H10, an antibody targeting N-terminal of H5 (A) h8A8 was able to

688

protect H5

689

antibody against H5N1 used as control; NA: no pH adjustment and no protease treatment. (B)

690

Western blotting with h8A8 against epitope mutants. 7H10 is a control Mab against the N

691

terminal of H5. Each lane was loaded with 20ul of viruses at HA titer of 28 (C) RG H5N1s

(CDC669) from low pH mediated degradation by protease K. 3H12: a HI

29

692

with individual mutations in either the conformational or linear epitope were tested in

693

protease susceptibility assays with h8A8.

694

Figure 7. Inhibition of cell-cell fusion in H5 transfected Vero cells with h8A8. 3H12: a HI

695

antibody against H5N1 used as control. Wild type CDC669 and RG H5N1s with individual

696

mutations in either the conformational or linear epitope were tested.

697 698

Tables

699

Table 1 Hemagglutination Inhibition (HI) titers of m8A8 against different influenza viruses. a

700

Concentration of m8A8 at 500 ug/ml. Virus A/Hong Kong/156/97 A/Hong Kong/213/03 A/Vietnam/1203/04 A/muscovy duck/Vietnam/33/07 A/Indonesia/CDC594/06 A/Indonesia/CDC669/06 A/Indonesia/CDC1031/2007 A/Chicken/Indonesia/TLL101/06 A/Duck/Indonesia/TLL102/06 A/turkey/Turkey1/05 A/Nigeria/6e/07 A/muscovy duck/Rostovon Don/51/07 A/Jiangsu/2/07 A/Anhui/1/05 A/Vietnam/HN31242/07 A/goose/Guiyang/337/06 A/chicken/Shanxi/2/06 A/chicken/Henan/12/04 A/Puerto Rico/8/34 A/Chicken/Malaysia/02 A/Netherlands/219/03 A/Chicken/Malaysia/98

H5 Clade/Subtype 0 1.0 1.0 1 2.1.2 2.1.3 2.1.3.2 2.1 2.1 2.2.1 2.2 2.2 2.3 2.3 2.3 4 7 9 H1N1 H3N2 H7N7 H9N2

HI a 128 256 256 128 512 512 256 256 512 256 256 128 256 512 256 128 256 128