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Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor 1

Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses 2

3

Xiaoquan Wanga, Natalia A. Ilyushinab, Vladimir Y. Lugovtseva, Nicolai V. Bovinc, Laura K. 4

Couzensa, Jin Gaoa, Raymond P. Donnellyb, Maryna C. Eichelbergera*, and Hongquan Wana* 5

6

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug 7

Administration, Silver Spring, Maryland, USAa; Division of Biotechnology Research and 8

Review II, Center for Drug Evaluation and Research, Food and Drug Administration, Silver 9

Spring, Maryland, USAb; Shemyakin Institute of Bioorganic Chemistry, Moscow, Russiac 10

11

Running title: Antigenic Phenotype of A(H3N2)v Virus 12 13 14

*Address correspondence to Maryna C. Eichelberger, maryna.eichelberger@fda.hhs.gov and 15

Hongquan Wan, hongquan.wan@fda.hhs.gov. 16

17

18

19

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Abstract word count: 249 21

Importance word count: 142 22

Text word count: 5595 23

JVI Accepted Manuscript Posted Online 2 November 2016J. Virol. doi:10.1128/JVI.01512-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 24

Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the US 25

in recent years, originated from human seasonal H3N2 viruses that were introduced into North 26

American swine in the mid-1990s, but are antigenically distinct from both the ancestral and 27

current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), 28

and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular 29

basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses 30

containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and 31

probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition 32

assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic 33

site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 34

156, 158, 189 and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype 35

to that of BJ/92. Mutations at residues 156, 157, 158, 189 and 193 of BJ/92 HA to amino acids 36

present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA 37

amino acid substitutions responsible for switching the antigenic phenotype also impacted HA 38

binding to sialyl receptors that are usually present in the human respiratory tract. Our study 39

demonstrates that antigenic site B residues play a critical role in determining both the unique 40

antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate 41

future surveillance and risk assessment of novel influenza viruses. 42

43

Keywords: A(H3N2)v, influenza virus, hemagglutinin, antigenic phenotype, amino acid44

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IMPORTANCE 45

Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human 46

infections in multiple states in the US since 2009. Most cases have been children who had 47

contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 48

viruses that were introduced into the US swine population in the mid-1990s, but are different 49

from both these 1990s ancestral viruses and current circulating human seasonal H3N2 strains in 50

terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In 51

this study, we identified amino acids in antigenic site B of the surface glycoprotein 52

hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral 53

H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, 54

suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 55

variants which pose a threat to public health. 56

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INTRODUCTION 57

Swine serve as an important intermediate host for influenza viruses to adapt, evolve and 58

acquire transmissibility to humans. The H1N1 pandemic in 2009 is a reminder that swine-origin 59

viruses represent an authentic public health threat (1). Influenza A H3N2 variant [A(H3N2)v] 60

viruses from swine have also caused human infections in multiple states in the US since 2009 (2-61

4), with more than 300 laboratory-confirmed cases in 2011 alone (5, 6). Most of the A(H3N2)v 62

cases have been children who had contact with swine at agricultural fairs (5, 7, 8). Fortunately 63

the symptoms of A(H3N2)v infection were generally mild, although some patients had to be 64

hospitalized, and one case was fatal (9). It is estimated that only 1 out of approximately 200 65

A(H3N2)v human infections are laboratory-confirmed; therefore, the number of actual human 66

cases might be much greater than that of reported cases (10). 67

A(H3N2)v viruses originated from human seasonal H3N2 strains that were introduced 68

into North American swine in the mid-1990s (11-13). After multiple reassortment events in 69

swine, these H3N2 viruses acquired a triple-reassortant internal gene (TRIG) cassette, containing 70

internal genes from avian, swine and human strains (11, 14-16). Outbreaks of TRIG-H3N2 virus 71

infections in US swine were first reported in a few states in 1998, and have spread to many other 72

states (11) and Canada (17, 18). The current circulating swine H3N2 viruses in North America, 73

together with A(H3N2)v viruses, were termed “lineage IV” based on the phylogenetic analysis 74

with earlier swine H3N2 isolates (13, 17-19). The majority of A(H3N2)v isolates since 2011 75

have acquired the matrix gene from the 2009 pandemic H1N1 virus (2, 20) , which has enhanced 76

the replication and transmission of A(H3N2)v viruses in swine (21, 22), potentially increasing 77

the risk of human infection. 78

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A(H3N2)v isolates are antigenically distinct from recent human seasonal H3N2 viruses 79

as well as H3N2 viruses that circulated in humans during the period when they are thought to 80

have been introduced into swine (2, 3, 19). Although amino acids close to the receptor binding 81

site (RBS) in hemagglutinin (HA) are associated with antigenic evolution of human seasonal 82

H3N2 and swine H3N2 viruses (23, 24), the specific residues that explain why swine-origin 83

A(H3N2)v viruses are antigenically distinct from the human strains have not been identified. 84

This information is important for our understanding of the emergence of novel antigenic 85

influenza strains that pose a threat to public health. In this study, we identified amino acids in 86

HA of A/MN/11/2010 (MN/10), the A(H3N2)v vaccine candidate, that provide the molecular 87

basis for the antigenic difference between A(H3N2)v and a proposed ancestral human seasonal 88

H3N2 strain, A/Beijing/32/1992 (BJ/92). We found that substitutions of these amino acids 89

impact receptor specificity, suggesting the antigenic differences may be the result of host 90

adaptation in addition to immune selection. 91

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MATERIALS AND METHODS 93

Wild-type (wt) viruses and ferret sera. A(H3N2)v virus A/Minnesota/11/2010 (MN/10) and 94

seasonal H3N2 virus A/Beijing/32/1992 (BJ/92) were grown in 11-day old embryonated 95

specific-pathogen free (SPF) chicken eggs and stored at -70°C until use. Ferret sera against 96

MN/10 and BJ/92 were generated by intranasal infection of ferrets with MN/10 or BJ/92 virus 97

following protocols approved by the Center for Biologics Evaluation and Research (CBER) 98

Animal Care and Use Committee. Briefly, ferrets were anesthetized with a mixture of ketamine 99

and xylazine intramuscularly, and inoculated intranasally with 107 median tissue culture 100

infectious doses (TCID50) of MN/10 or BJ/92 virus diluted in 1 ml PBS, 0.5 ml/nostril. Ferrets 101

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were bled 3 weeks after the infection, and the sera were stored at -30 °C. Sera collected from 4 102

ferrets, 2 for MN/10 antiserum and 2 for BJ/92 antiserum, were used in the studies. 103

Site-directed mutagenesis. Nucleotide changes corresponding to single or multiple amino acid 104

mutations were introduced into the HA gene in pHW2000 plasmid with QuickChange® multi 105

site-directed mutagenesis kit (Stratagene, La Jolla, CA). The resulting plasmids were sequenced 106

to verify the presence of introduced mutations and the absence of additional, unwanted 107

mutations. 108

Reverse genetics. Reassortant MN/10 and BJ/92 viruses that bear the wt or mutant HA and NA 109

genes of MN/10 or BJ/92, and the internal genes of an H1N1 virus A/Puerto Rico/8/1934 (PR8), 110

were generated as previously reported (25, 26). Briefly, plasmids (1 μg each) containing each of 111

the 8 virus genes were transfected into a mixture of 293T human embryonic kidney cells and 112

Madin-Darby canine kidney (MDCK) cells. The transfection mixture was replaced with OPTI-113

MEM I medium (Invitrogen, Grand Island, NY) after 6 h of incubation at 37°C. OPTI-MEM I 114

medium supplemented with 1 μg/ml trypsin was added 24 h later. At 48 to 72 h post transfection, 115

the culture supernatant was collected and propagated in 11-day old embryonated SPF chicken 116

eggs. 117

Hemagglutination inhibition (HI) assay. The antigenic phenotypes of wt and reassortant 118

viruses were determined by performing HI assays. Briefly, ferret sera were treated overnight at 119

37 °C with receptor-destroying enzyme from Vibrio cholerae (Denka Seiken, Tokyo, Japan), and 120

then heat inactivated at 56 °C for 30 min. After dilution with PBS to 1:10, the sera were 121

absorbed with packed turkey red blood cells to remove nonspecific inhibitors, and then serially 122

diluted before mixing with 4 hemagglutination units of virus and 0.5% turkey red blood cells. 123

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Two independent HI assays were performed for each virus and the geometric mean titers were 124

calculated. 125

Receptor-binding assay. The affinity of each virus for biotinylated 6′-sialylglycopolymers was 126

measured in a direct binding assay as described previously (27). Briefly, plates were coated with 127

4-8 hemagglutination units of each virus at 4 °C for 16 h followed by washing with 0.05% 128

Tween 20 in phosphate-buffered saline (PBS-T). After the addition of biotinylated-129

sialylglycopolymer in PBS supplemented with 0.02% Tween 20, 0.02% bovine serum albumin 130

(BSA), and 3 μM oseltamivir carboxylate, plates were incubated at 4 °C for 1 h. Plates were then 131

washed with cold PBS-T and incubated with streptavidin-peroxidase (Sigma-Aldrich, St. Louis, 132

MO) at 4 °C for 1 h. After washing, TMB substrate solution (KPL, Gaithersburg, MD) was 133

added and the reaction was stopped with TMB stop solution (KPL, Gaithersburg, MD). Optical 134

density was determined at 450 nm with a Synergy 2 multimode microplate reader (BioTek 135

Instruments, Winooski, VT). The dissociation constants (Kd) values were determined by 136

Scatchard plot analysis; Kd was defined as the sialic acid (µM of Neu5Ac) concentration at ½ 137

Amax, where Amax represents maximum absorbance. The reported data represent the mean of at 138

least four individual experiments for each virus. 139

Generation of monoclonal antibodies (MAbs) against the HA of MN/10 virus. A panel of 140

HA-specific mouse MAbs was used in the analysis of antibody binding to viruses. Hybridomas 141

that secrete MAbs were generated through immunizing BALB/c mice (The Jackson Laboratory, 142

Bar Harbor, ME) with inactivated MN/10 virus and fusing splenocytes from an immunized 143

mouse with Sp2/0 cells. The HA specificity of MAbs was confirmed by ELISA using 293T cells 144

transfected with an HA-expressing plasmid (pCAGGS-MN/10 HA) as antigen (28). Selected 145

hybridomas were cultured in CELLine device (BD Biosciences, San Jose, CA) and the MAbs 146

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purified using protein G columns (GE Healthcare, Uppsala, Sweden). The key residues in HA for 147

antibody binding were identified by sequencing the HA gene of escape mutants of MN/10 virus, 148

which were selected in 11-day old embryonated SPF chicken eggs in the presence of MAb (28). 149

Antibody-binding assay. The binding of viruses by MAbs was measured by ELISA. Briefly, 150

purified wt and mutant viruses were coated onto Immulon 2HB Microtiter® plates (Thermo 151

Scientific, Rochester, NY) at 1 µg/well. The virus-coated plates were blocked with 1% BSA in 152

PBS and then incubated with each MAb (1 µg/ml) in 1% BSA-PBST. After an additional 153

incubation with peroxidase-conjugated goat-anti-mouse IgG (Sigma-Aldrich, St. Louis, MO), the 154

signal was developed using o-phenylenediamine dihydrochloride (OPD) as substrate. The 155

reaction was stopped with 1N H2SO4 and OD490 values were read. The OD490 values of mutant 156

viruses and wt BJ/92 were normalized to those of wt MN/10. Two independent assays were 157

performed and each was run in duplicate wells. 158

Gene sequencing and alignment. The HA gene of each virus was sequenced to confirm the 159

expected mutations. Viral RNA was extracted from infectious allantoic fluid with an RNeasy® 160

mini kit (Qiagen, Valencia, CA). cDNA synthesis and PCR were performed as previously 161

described (29) to amplify the HA gene. PCR products were sequenced at the Facility for 162

Biotechnology Resources, CBER, FDA. DNA and amino acid sequences were analyzed with 163

DNASTAR Lasergene software version 13 (DNASTAR, Inc., Madison, WI). 164

Statistical analysis. The Kd values were compared by analysis of variance (ANOVA). 165

Probability values ≤ 0.05 indicate statistically significant differences. Associations between HI 166

titers and receptor binding affinity were evaluated using Spearman correlation test. 167

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RESULTS 168

Antigenic difference between A(H3N2)v MN/10 and seasonal H3N2 virus BJ/92. To 169

determine the molecular basis for the antigenic difference between A(H3N2)v and seasonal 170

H3N2 viruses, an A(H3N2)v virus, MN/10, and a seasonal H3N2 virus BJ/92, were used as 171

prototype viruses in this study. MN/10 is a vaccine candidate for potential A(H3N2)v outbreaks. 172

BJ/92 was selected because it is genetically closely related to the North American swine H3N2 173

viruses first isolated in the late 1990s (11). BJ/92 was also used as a vaccine strain during the 174

1993-1994 influenza season. We performed HI assays to determine antigenic phenotype with 175

two sets of ferret sera (each included MN/10 antiserum and BJ/92 antiserum). Since a similar 176

pattern of inhibition was observed with both sera, only one set of data is reported. In HI assays, 177

ferret sera against MN/10 and BJ/92 inhibited the homologous virus with HI titers of 453 and 178

640, respectively; however, no inhibition was observed against the heterologous virus (HI titers 179

<10) (Table 1), confirming the antigenic difference between these two viruses. 180

Amino acids in antigenic site A have little impact on the antigenic phenotype of MN/10. 181

Because HA is the target of antibodies in HI assay, the difference of HI titers is a good indicator 182

of amino acid variations in the HA. Indeed, there are 51 amino acid differences between the 183

MN/10 and BJ/92 HAs, with 38 distributed throughout HA1 (Fig. 1). Many of these variations 184

are within known antigenic domains, e.g., antigenic sites A, B, C, D and E (18, 30-32). Among 185

these, 4 are in antigenic site A, whereas 10 are in antigenic site B. To determine which of these 186

amino acid variations is responsible for the observed antigenic difference, we focused on 187

residues in antigenic sites A and B because antibodies against these two sites are the most 188

effective at inhibiting hemagglutination. Reassortant MN/10 and BJ/92 viruses with the PR8 189

internal gene constellation were generated by reverse genetics, and residues in antigenic sites A 190

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and B in the HA of reassortant MN/10 were mutated to those in BJ/92 or vice versa. The wt and 191

mutant viruses were tested in HI assays to identify amino acid changes that reduced inhibition by 192

the homologous antiserum but increased inhibition by the heterologous antiserum. Mutations 193

that caused ≥ 4-fold reduction in HI titers of the homologous antiserum and a ≥ 4-fold increase in 194

HI titers of the heterologous antiserum were deemed to be determinants for the antigenic 195

phenotype. 196

As shown in table 1, reassortant MN/10 viruses, each bearing one of the single residue 197

substitutions in antigenic site A to those present BJ/92 HA, i.e., Q122N, S124D, S135G and 198

R140K, were inhibited by the homologous MN/10 antiserum at HI titers of 320 or 453, similar to 199

that against wt MN/10 (HI titer 453). Consistently, these viruses were not inhibited at all, or only 200

slightly inhibited by BJ/92 antiserum (HI titers ≤10). A reassortant MN/10 virus possessing all of 201

these 4 MN/10 to BJ/92 mutations was still effectively inhibited by MN/10 antiserum (this virus 202

was abbreviated as 122/124/135/140, and other mutants with more than one residue mutation 203

were abbreviated similarly throughout the text), and was only weakly inhibited by BJ/92 204

antiserum, at an HI titer of 20. These data indicate that amino acid substitutions in antigenic site 205

A do not explain the observed antigenic difference between MN/10 and BJ/92. 206

Amino acids in antigenic site B determine the antigenic phenotype of MN/10. We next 207

examined whether substitutions in antigenic site B are associated with the antigenic phenotype of 208

A(H3N2)v viruses. Residues 155, 156, 158, 189 and 193 in antigenic site B of MN/10 HA were 209

examined because these residues are commonly associated with antigenic drift of human 210

seasonal and swine influenza viruses (23, 24, 33, 34). Reassortant MN/10 viruses bearing each of 211

the 5 single mutations from MN/10 to BJ/92, Y155H, N156K, N158E, K189R or K193S, were 212

inhibited by MN/10 antiserum at slightly lower HI titers (160, 226 or 320) compared to wt 213

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MN/10; however, none of these single mutant viruses recovered sufficient reactivity with BJ/92 214

antiserum, i.e., HI titers of BJ/92 antiserum were all below the detection limit, 10, except a titer 215

of 14 against the N158E mutant virus. In contrast to these single residue mutants, the 216

combination of these 5 mutations in a reassortant MN/10 virus (155/156/158/189/193) restored 217

inhibition by BJ/92 antiserum, with an HI titer of 226, which is ˂ 4-fold difference from that of 218

wt BJ/92, 640. Mutations of all of these 5 residues also resulted in a substantial loss of inhibition 219

by MN/10 antiserum, with a >16-fold reduction in HI titer compared to the wt MN/10. These 220

data demonstrate that amino acid mutations in antigenic site B account for the unique antigenic 221

phenotype of MN/10 and its antigenic difference from BJ/92. 222

Minimal number of residues in HA that switch the MN/10 antigenic phenotype to that of 223

BJ/92. Since our data indicated that the residue changes in antigenic site B underlie the antigenic 224

difference between MN/10 and BJ/92, we performed HI assays to determine the minimal 225

determinant that is associated with the antigenic difference. The reactivity of MN/10 and BJ/92 226

antisera were tested against reassortant MN/10 viruses containing various combinations of 227

mutations at the 5 antigenic site B residues (155, 156, 158, 189 and 193), including almost all of 228

the possible double, triple and quadruple mutations. While some of the double and triple 229

mutants, e.g., 155/156, 155/193, 156/189/193, were inhibited at HI titers < 4-fold difference from 230

that of wt MN/10 (i.e., HI titers 160 and 320 versus 453 of the wt MN/10), most of the double 231

and triple mutants had > 4-fold decrease in HI titers, i.e., they were inhibited by MN/10 232

antiserum at HI titers of 57-113. Moreover, while some double and triple mutants, e.g., 155/189 233

and 156/189, gained no reactivity with BJ/92 antiserum, some other mutants, such as 234

156/189/193 and 158/189/193, exhibited substantially enhanced inhibition by BJ/92 antiserum 235

(HI titers between 40 and 113, respectively). The decrease in HI titers against the homologous 236

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MN/10 antiserum was accompanied by the increase in HI titers against the heterologous BJ/92 237

antiserum. 238

The MN/10 antiserum had ≥ 8-fold lower HI titers against all of the quadruple mutants, 239

demonstrating that these mutants were antigenically different from the wt MN/10 virus. The 240

impact of quadruple mutations was also evident in the reactivity pattern of BJ/92 antiserum- all 241

of the quadruple mutants, except 155/156/158/189, became susceptible to inhibition by BJ/92 242

antiserum, with HI titers ≥ 80. One of the quadruple mutants, 156/158/189/193, was 243

antigenically similar to wt BJ/92, with < 4-fold HI titer difference from wt BJ/92 virus: HI titers 244

of 226 and 640, respectively, with BJ/92 antiserum (Table 1 and Fig. 3). Thus, a minimum of 4 245

amino acid substitutions at positions 156, 158, 189 and 193 in antigenic site B switched the 246

antigenic phenotype of MN/10 to that of BJ/92. 247

Amino acid 157 plays a role in determining the antigenic difference between MN/10 and 248

BJ/92. Our results showed that mutants 156/158/189/193 and 155/156/189/193 were 249

antigenically similar to BJ/92; however, the HI titers of BJ/92 antiserum against both mutants 250

were still > 2-fold lower than the wt BJ/92 virus (226 versus 640). Therefore, we next tested 251

whether additional mutations, including those single mutations that did not have a profound 252

impact on the antigenic phenotype, could enhance the inhibition by BJ/92 antiserum. The L157S 253

mutation was examined in particular because of its structural proximity to residues 156, 158, 189 254

and 193 (Fig. 2). While the L157S mutation alone did not alter the antigenic phenotype of 255

MN/10, introduction of this change into mutants 156/158/189/193 and 155/156/158/189/193 256

enhanced inhibition by BJ/92 antiserum, reaching HI titers very similar to that against the wt 257

BJ/92 virus (453 versus 640; table 1 and Fig. 3). The addition of S135G to 155/156/158/189/193 258

virus also enhanced the inhibition by BJ/92 antiserum (Table 1). These findings indicate that 259

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additional substitutions in the HA can enhance binding of polyclonal antibodies and thereby 260

contribute to the antigenic difference between A(H3N2)v and seasonal H3N2 viruses. 261

To confirm the importance of residues 156, 158, 189 and 193 within antigenic site B for 262

inhibition by BJ/92 antiserum, we rescued a reassortant BJ/92 virus that carries HA and NA 263

genes of BJ/92 and the internal genes of PR8, with HA residues 156, 158, 189 and 193 mutated 264

to those present in MN/10 HA. This mutant BJ/92 reassortant virus, abbreviated as 265

BJ156/158/189/193, had dramatically reduced sensitivity to BJ/92 antiserum, with HI titer of 20 266

compared to 640 of wt BJ/92, confirming the key role of these amino acids in recognition of 267

BJ/92-specific antibodies; however, this virus was only weakly inhibited by MN/10 antiserum, 268

with an HI titer of 40. Interestingly, when S157 in this reassortant BJ/92 virus was mutated to L, 269

which is present in MN/10 HA, the HI titer of MN/10 antiserum against the reassortant 270

increased, resulting in < 4-fold difference from wt MN/10 (Table 1 and Fig. 3), thereby 271

switching the BJ/92 antigenic phenotype to that of MN/10. These results further highlight the 272

critical role of amino acids in antigenic site B, including residue 157, in defining the antigenic 273

difference between MN/10 and BJ/92 viruses. 274

HA amino acid substitutions responsible for switching the antigenic phenotype affect 275

receptor specificity. Since antigenic site B is in close proximity to the RBS, we investigated the 276

effect of HA amino acid substitutions at positions 156, 157, 158, 189 and 193 that are 277

responsible for switching antigenic phenotype, on receptor specificity of the virus. We measured 278

the affinities of MN/10, BJ/92 and five mutants, 156/158/189/193, 155/156/158/189/193, 279

156/157/158/189/193, BJ156/158/189/193, and BJ156/157/158/189/193 for synthetic 280

Neu5Acα2,6-glycopolymers (Table 2). The binding pattern to the major analogs of human 281

receptors 6ʹSL, 6ʹSLN, sulfated 6-Su-6ʹSLN and the surrogate of human receptors Neu5AcBn 282

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(35), and was identical among all viruses tested (see table 2 for structures and abbreviations of 283

sialylglycopolymers). The binding of MN/10 and BJ/92 to 9NAc-6ʹSLN and 6ʹSLN(Gc), glycans 284

that are represented only in swine respiratory epithelial cells (35-37), was significantly distinct 285

and independent of amino acids 156, 157, 158, 189 and 193 because viruses with mutations at 286

these sites retained the parental HA binding pattern. Despite the fact that the glycan SiaTn is 287

rarely found in the human respiratory tract (38), MN/10 and BJ/92 both bound this glycan with 288

high affinity, although binding of the MN/10 virus was significantly greater (Kd < 0.1 µM of 289

Neu5Ac compared to BJ/92 that had a Kd 1.5 ± 0.4 µM of Neu5Ac, P < 0.05, table 2). 290

Interestingly, the mutations at amino acids that we identified as determinants of the 291

antigenic phenotype contributed to the switch in binding affinity. Binding of MN/10 towards 292

SiaTn and Neu5Ac6Gal was significantly reduced when residues 156, 158, 189 and 193 were 293

mutated to those in BJ/92 (P < 0.05). The binding affinity of BJ/92 significantly increased when 294

these 4 residues were replaced with amino acids from MN/10. A similar switch in binding 295

affinity was observed for glycans YDS, 6-SiaTF, and swine specific receptor Neu5Gc-Tn (37) in 296

wt MN/10 and MN/10-like mutants. However, unlike binding to previously mentioned glycans, 297

the amino acid at position 157 of HA had a substantial impact on the affinity of the BJ/92 virus. 298

For example, introduction of S157L mutation into mutant BJ156/158/189/193 resulted in 299

acquisition of moderate binding to Neu5Gc-Tn and 6-SiaTF receptors (i.e., the increase in 300

receptor binding affinity was dependent on amino acid 157, table 2, P ˂ 0.05). In addition, there 301

was a significant positive correlation between HI titers of MN/10 antiserum and binding to 2 302

similar sialylglycopolymers, SiaTn and 6-SiaTF, which are rarely found in humans (38, 39). 303

Spearman correlation coefficients were 0.78, P=0.049 and 0.80, P=0.048, respectively (Fig. 4). 304

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Our data demonstrated that amino acids which are critical antigenic determinants impact the 305

affinity of A(H3N2)v viruses for some minor human glycans. 306

HA amino acid substitutions responsible for switching the antigenic phenotype have 307

profound impact on antibody binding. Finally we investigated whether differences in HI titer 308

were due to a direct effect of antibody binding. We selected 8 mouse MAbs against the MN/10 309

HA and measured their binding to some of the MN/10 and BJ/92 mutants by ELISA. Six of these 310

MAbs inhibited MN/10 virus in the HI assay (Table 3). This observation and the sequencing data 311

of the escape mutants indicate that they target epitopes in antigenic sites A and B in the globular 312

head of the HA. The remaining 2 antibodies (HA13 and HA59) do not inhibit hemagglutination, 313

implying that they bind antigenic sites C, D, E or the stalk of HA. MAb binding to wt MN/10, wt 314

BJ/92 and 5 mutants that exhibited substantial antigenic change (156/158/189/193, 315

155/156/158/189/193, 156/157/158/189/193, BJ156/158/189/193 and BJ156/157/158/189/193), 316

was tested in the ELISA (Fig. 5). MN/10 with a single mutation at residue 157 to that in BJ/92 317

(L157S) was also included. All of the 8 MAbs reacted well with wt MN/10, with OD490 values 318

between 1.9 and 2.5 versus ˂0.1 for the negative control (data not shown). The mutations that 319

changed the antigenic phenotype of MN/10 to that of BJ/92 caused dramatic loss of binding by 320

some of the MAbs that possess HI activity. For example, relative to wt MN/10, the 321

156/158/189/193 virus had significantly lower level of binding by MAbs HA20, HA25 and 322

HE42, and completely lost binding by MAb HB27; mutant 155/156/158/189/193 was not bound 323

by antibodies HA20, HA25, HB27, HE42, and had reduced binding by MAb HB1. Antibody 324

HB67 targets antigenic site A (Table 3), therefore its reactivity with MN/10 virus was not 325

significantly changed by the tested mutations, which are all within antigenic site B. Weak 326

binding of HB67 to BJ/92 was observed, suggesting some level of conservation in antigenic site 327

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A of MN/10 and BJ/92. Only MAbs HA13 and HA59 bound all MN/10 mutants (Only the 328

HA13 data are shown in Fig. 5). While none of the MAbs reacted strongly with wt BJ/92, 329

binding by most of these antibodies was observed when BJ/92 HA residues were mutated to 330

those in MN/10 (Fig. 5). These data indicate that the amino acid mutations introduced into the 331

HA of MN/10 and BJ/92 directly impact antibody binding to HA, which is likely the major 332

reason for the observed antigenic switch. 333

Interestingly, while the L157S mutation alone did not impact the binding by the tested 334

MAbs, the addition of L157S in mutant 156/158/189/193 decreased the binding of antibody HB1 335

to the resultant virus 156/157/158/189/193 (Fig. 5). More interestingly, while 336

BJ156/158/189/193 was not recognized by HB1 in our ELISA, BJ156/157/158/189/193, with 337

S157 mutated to L in MN/10, reacted with HB1 as effectively as wt MN/10 virus. This finding is 338

consistent with the observation that the MN/10 antiserum has a 4-fold higher HI titer with 339

BJ156/157/158/189/193 than BJ156/158/189/193 (Table 1), and implies that residue 157, 340

together with other residues (e.g., 156, 158, 189, 193) is a critical determinant of the antigenic 341

phenotype. 342

343

DISCUSSION 344

H3N2 influenza A viruses are endemic in the US swine population. Some of these swine 345

H3N2 viruses, e.g., the H3N2 variants that have been transmitted to humans in the past years, 346

pose a potential threat, particularly to children. Serological studies with human serum samples 347

demonstrate that while a significant proportion of adolescent and young adults have cross-348

reactive antibodies against A(H3N2)v viruses, young children lack such preexisting immunity 349

(40, 41). Investigations in the ferret model suggest that vaccination with seasonal trivalent 350

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inactivated influenza vaccines do not provide protection against transmission of A(H3N2)v virus 351

(42, 43). These findings highlight the antigenic difference between circulating H3N2 and the 352

swine-origin viruses and the need to evaluate and approve A(H3N2)v vaccines. In this study, we 353

identified amino acids in the HA of MN/10, the A(H3N2)v vaccine candidate, that explain why 354

this virus is antigenically distinct from an ancestral human seasonal H3N2 strain, BJ/92. In 355

addition, we demonstrated that the amino acid substitutions that confer the antigenic phenotype 356

impact both receptor specificity and antibody binding. Our findings provide additional evidence 357

that residues near the HA RBS, especially those in antigenic site B, play significant role in the 358

antigenic evolution of H3N2 viruses (23, 24) . 359

Seasonal H3N2 viruses experience antigenic drift, which necessitates frequent updates of 360

H3N2 candidate viruses for seasonal influenza vaccines (24, 34). Antigenic drift of H3N2 361

viruses is often due to single or double amino acid mutations at several positions in the HA, e.g., 362

145, 155, 156, 158, 189 and 193 (24). A similar antigenic drift mechanism was also observed in 363

H1N1 and H5N1 viruses (44, 45). However, evolution over longer periods of time may result in 364

a larger number of amino acid changes in antigenic sites. For example, 5 amino acid 365

substitutions are responsible for the antigenic divergence from the 1968 to 2002 H3N2 viruses 366

(A/Hong Kong/1/1968 to A/Fujian/411/2002 (24). Consistent with this observation, our data 367

from this study indicate that at least 4-5 amino acid mutations are needed to switch the antigenic 368

phenotypes between A(H3N2)v and the ancestral seasonal H3N2 viruses. This may be due to 369

multiple reasons. First, it has been ~20 years since the introduction of human H3N2 viruses into 370

the US swine population. Multiple antigenic drifts could have occurred during the evolution in 371

such a long period. Furthermore, swine represent a very different evolutionary host from 372

humans in terms of physiology, life span, vaccination regime, etc. Some of the residues that are 373

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associated with the antigenic difference might also be the result of host adaptation. Indeed, the 374

residues critical for the antigenic difference between MN/10 and BJ/92 are also associated with 375

the receptor binding property of the viruses. Taken together, both the long term antigenic 376

evolution and host adaptation might have determined the molecular basis for the observed 377

antigenic difference between A(H3N2)v and seasonal H3N2 viruses. These changes are not as 378

simple as the antigenic drift seen in human seasonal influenza viruses, which is often caused by a 379

single amino acid mutation in the HA. 380

The amino acid residues identified in the present study as critical for the antigenic 381

deference between A(H3N2)v and seasonal H3N2 viruses are all located near the RBS in HA, 382

which is in agreement with the observations of other studies that examined H3N2 antigenic 383

evolution (19, 23, 24). Our study indicated that the amino acid mutations which switched the 384

antigenic phenotype of MN/10 and BJ/92 influenced receptor binding. Moreover, these 385

mutations have a profound direct impact on binding of the HA by mouse MAbs. Consistent with 386

this finding, mutations at these residues have also been found to impact the binding by human 387

MAbs (46). Taken together, the antigenic difference between MN/10 and BJ/92 is likely the 388

result of changes in receptor binding avidity as well as antibody selection. Interestingly, all of 389

the identified critical residues are located within antigenic site B, adding evidence that residue 390

substitutions in antigenic site B more frequently impact antibody recognition than those in 391

antigenic site A. It was somewhat surprising that antigenic site B is the primary target of both 392

MN/10 and BJ/92 ferret antisera, since there are a number of additional amino acid differences 393

between these HAs. The fact that antigenic site A of A(H3N2)v viruses also included residue 394

substitutions suggests that selective pressure is present on this antigenic site during the evolution 395

of H3N2 in swine. A very recent study reported the isolation of human antibody H3v-41, which 396

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binds antigenic site A (46), demonstrating that immunity against antigenic site A was induced in 397

humans vaccinated against A(H3N2)v. In our study, one of the escape mutants selected by 398

mouse MAb HE42 bears an N to K mutation at residue 145, suggesting that antigenic site A is 399

also targeted by the mouse immune response. The reason why ferret antisera identify residues 400

within antigenic site B as the major determinants for the antigenic difference between MN/10 401

and BJ/92 viruses deserves further investigation. 402

Ferret antisera are routinely used in HI assays to characterize the antigenic phenotype of 403

influenza viruses. Since these sera are collected from ferrets recovering from primary influenza 404

virus infection, it is likely that the specificity and avidity of antibodies may differ from human 405

antisera. For instance, serological assays with human antiserum samples can discriminate 406

between K166 and Q166 in the HA of H1N1 viruses; this difference was not detected by ferret 407

antisera (47). The K116Q mutation has been proposed to be one of the reasons for the increased 408

morbidity and mortality of influenza among middle-aged people during the 2013-2014 influenza 409

season (47). In light of this observation, a comprehensive analysis of data generated with both 410

ferret and human antisera might facilitate a better understanding of influenza virus evolution as 411

well as decisions to update influenza vaccines. 412

Our results show that residue 157, which is not often reported to be involved in antigenic 413

evolution, also played a role in determining the antigenic difference between A(H3N2)v and 414

seasonal H3N2 viruses. Our ELISA data demonstrated that residue 157, in combination with the 415

neighboring and adjacent residues, e.g., 156, 158, 189 and 193, has a direct impact on the 416

binding of antibodies such as HB1 to HA. This residue was also observed to influence the 417

receptor-binding avidity of the BJ/92 mutant to certain glycans (e.g., Neu5Gc-Tn and 6-SiaTF). 418

Residue 157 is within the 150-loop that constitutes the RBS, and is in close proximity to other 419

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residues (e.g., those in 190-helix) that are critical for the antigenic phenotype, so it is not 420

surprising that it may also interact with antibodies and contribute to the avidity of HA when 421

binding to some glycans. Regardless of the reasons why particular amino acid changes were 422

selected during the evolution of MN/10, our results show that residues in antigenic site B, 423

especially those at positions 156, 157, 158, 189 and 193, deserve close monitoring in 424

surveillance and risk assessment of emerging H3N2 viruses. 425

Previous studies have shown that HA mutations can decrease HI antibody activity by 426

increasing the viral HA binding avidity to cell surface glycan receptors (48, 49). Our data 427

identified MN/10 and BJ/92 mutants that switched both their antigenic and receptor binding 428

phenotypes to that of the heterologous virus, suggesting that a change in receptor binding affinity 429

can impact antigenicity. Similarly, Hensley et al. (50) demonstrated a positive correlation 430

between receptor binding avidity and escape from polyclonal antibodies of H1 influenza viruses, 431

indicating that variations in receptor binding support the emergence of antibody-selected 432

mutants. Interestingly, our data indicate a significant positive correlation between HI titers of 433

MN/10 antiserum and binding to SiaTn and 6-SiaTF sialylglycopolymers. These results suggest 434

that changes in receptor binding properties correlate with antigenic structure (40), and therefore 435

both host adaptation and immune selection are likely to contribute to the antigenic differences 436

between A(H3N2)v and seasonal H3N2 viruses. 437

In conclusion, the results we present in this report confirm that A(H3N2)v viruses are 438

antigenically distinct from the ancestral seasonal H3N2 strains that circulated in the early 1990s, 439

and identify multiple amino acids in antigenic site B of HA are critical for this antigenic 440

difference. This is in agreement with previous observations that substitutions near the HA RBS 441

determine antigenic changes during influenza virus evolution. Our findings define the molecular 442

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basis for the unique antigenic phenotype of A(H3N2)v viruses, and may facilitate future 443

surveillance and risk assessment of novel influenza viruses. 444

445

FUNDING INFORMATION 446

This study was supported by intramural FDA funds and by RAS Presidium Grant 447

Molecular and Cell Biology to N.V.B. 448

449

ACKNOWLEDGEMENTS 450

We thank Robert G. Webster (St Jude Children’s Research Hospital, Memphis, TN) for 451

providing plasmids used to generate influenza viruses by reverse genetics. We are indebted to 452

staff of the Division of Veterinary Services, CBER, FDA, for excellent animal care and the 453

Facility for Biotechnology Resources, CBER, FDA for sequence analyses. 454

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50. Hensley SE, Das SR, Bailey AL, Schmidt LM, Hickman HD, Jayaraman A, Viswanathan K, Raman 606 R, Sasisekharan R, Bennink JR, Yewdell JW. 2009. Hemagglutinin receptor binding avidity drives 607 influenza A virus antigenic drift. Science 326:734-736. 608

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TABLE 1 HI titers of ferret antisera against mutant MN/10 and BJ/92 viruses 611 612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

Viruses Antigenic site

HI titera of ferret sera MN/10 BJ/92

wt MN/10 453 5 wt BJ/92 5 640 Q122Nb A 453 10 S124D A 453 10 S135G A 320 10 R140K A 320 5

122/124/135/140 A 320 20 Y155H B 226 5 N156K B 160 5 L157S B 320 10 N158E B 226 14 K189R B 320 5 K193S B 160 5 155/156 B 226 14 155/158 B 113 14 155/189 B 113 5 155/193 B 160 28 156/189 B 40 5 156/193 B 80 20 158/189 B 160 14 158/193 B 160 20 189/193 B 160 20

155/156/158 B 113 40 155/156/189 B 57 28 155/156/193 B 57 57 155/158/189 B 57 20 155/158/193 B 80 57 155/189/193 B 160 28 156/158/189 B 57 20 156/158/193 B 113 40 156/189/193 B 160 113 158/189/193 B 113 80

155/156/158/189 B 28 28 155/156/158/193 B 57 113 155/156/189/193 B 40 113 155/158/189/193 B 57 80 156/158/189/193 B 57 226

155/156/158/189/193 B 28 226 156/157/158/189/193 B 80 453

135/155/156/158/189/193 A and B 28 453 155/156/157/158/189/193 B 28 453

BJ156/158/189/193c B 40 20 BJ156/157/158/189/193 B 160 40

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aGeometric mean titer from two independent assays; 5 was assigned to titers <10 for calculation. 635

bReassortant viruses possessing HA and NA genes of MN/10 virus and the internal genes of PR8, 636

each containing a single residue change (e.g., Q122N) or a combination of changes, e.g., 122, 637

124,135 and 140 (abbreviated as 122/124/135/140), to that/those present in BJ/92 HA. 638

cReassortant viruses containing HA and NA genes of BJ/92 and the internal genes of PR8, with 639

residues at indicated positions mutated to those in MN/10 HA. 640

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TABLE 2 Effect of H3 HA amino acid substitutions responsible for switching the antigenic phenotype on receptor specificitya 641

Biotinylated sialylglycopolymers Viruses

wt MN/10 wt BJ/92 156/158/189/193 155/156/158/189/193 156/157/158/189/193 BJ156/158/189/193 BJ156/157/158/189/193

Similar binding compared to wt virus Neu5Acα2-OCH2C6H4 (Neu5AcBn) 0.1 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 Neu5Acα2-6Galβ1-4Glc (6ʹSL) 0.5 ± 0.1 1.1 ± 0.3 1.0 ± 0.3 0.8 ± 0.3 0.9 ± 0.3 0.7 ± 0.2 0.6 ± 0.2 Neu5Acα2-6Galβ1-4GlcNAc (6ʹSLN) 0.7 ± 0.3 1.2 ± 0.5 1.5 ± 0.6 0.7 ± 0.3 1.2 ± 0.4 1.6 ± 0.7 1.2 ± 0.4 Neu5Acα2-6Galβ1-4(6-O-Su)GlcNAc (6-Su-6ʹSLN) 0.5 ± 0.1 0.7 ± 0.1 0.7 ± 0.2 0.5 ± 0.1 0.7 ± 0.3 0.9 ± 0.3 0.8 ± 0.2

9-(AcN)-Neu5Acα2-6Galβ1-4GlcNAc (9NAc-6ʹSLN) 1.3 ± 0.5° ˃ 50* 1.0 ± 0.3° 0.5 ± 0.2° 1.0 ± 0.4° ˃ 50* ˃ 50*

Neu5Gcα2-6Galβ1-4GlcNAc (6ʹSLN(Gc)) 15.0 ± 4.1° ˃ 50* 22.3 ± 10.8° 13.2 ± 6.1° 25.6 ± 7.0° ˃ 50* ˃ 50*

Different binding compared to wt virus Neu5Acα2-6GalNAcα (SiaTn) <0.1° 1.5 ± 0.4* 1.0 ± 0.4* 0.9 ± 0.3* 0.9 ± 0.3* 0.4 ± 0.1° 0.4 ± 0.1° Neu5Acα2-6Galβ (Neu5Ac6Gal) 0.9 ± 0.4° 4.5 ± 0.8* 4.7 ± 0.7* 3.9 ± 0.6* 4.9 ± 0.3* 1.8 ± 0.4° 1.3 ± 0.3°

Neu5Acα2-6Galβ1-4GlcNAc-Man- GlcNAc-GlcNAcNeu5Acα2-6Galβ1-4GlcNAc-Man (YDS)

0.6 ± 0.2° 3.6 ± 0.5* 3.0 ± 0.9* 2.2 ± 0.4* 2.8 ± 0.4* 4.2 ± 1.1* 2.9 ± 0.7*

Neu5Gcα2-6GalNAcα (Neu5Gc-Tn) 0.6 ± 0.3° ˃ 50* 11.3 ± 4.0*,° 10.6 ± 2.5*,° 16.5 ± 5.2*,° ˃ 50* 15.0 ± 2.9*,° Neu5Acα2-6(Galb1-3)GalNAcα (6-SiaTF) 5.0 ± 2.2° ˃ 50* ˃ 50* ˃ 50* ˃ 50* ˃ 50* 19.5 ± 1.9*,° Neu5Acα2-6(Neu5Aca2-3)GalNAcα (3,6-SiaTn) ˃ 50 ˃ 50 ˃ 50 ˃ 50 ˃ 50 6.3 ± 3.7*,° 3.3 ± 2.0*,°

aThe dissociation constants (Kd, µM of Neu5Ac) of virus complexes with synthetic sialylglycopolymers are shown. Lower Kd values indicate 642

stronger binding. The values represent the mean ± standard deviation (SD) of at least 4 independent experiments. °P < 0.05, compared to the 643

values for wt BJ/92 virus by one-way ANOVA; *P < 0.05, compared to the values for wt MN/10 virus by one-way ANOVA. 644

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TABLE 3 Mouse MAbs used for antibody-binding assay 645

646

647

648

649

650

651

652

653

aMeasured with 1mg/ml of each MAb. MN/10 antiserum collected from the mouse used 654

for generation of the HA-specific MAbs was used as a positive control. 655

bnd, not done. 656

cThese mutations are present in different mutants selected by the same MAb. 657

dThe mutant possesses a deletion of 2 amino acids, either 156NL157 or 157LN158, from 658

the wt sequence 155YNLNYK160. 659

MAb Isotype HI titera Mutation in HA of escape mutants

HA13 IgG1 <10 ndb

HA20 IgG1 10240 K160T or N165Kc HA25 IgG1 5120 Y159D

HA59 IgG1 <10 nd

HB1 IgG2a 5120 155YN- -YK160d HB27 IgG2a 10240 Y159D or 155YN- -YK160

HB67 IgG2a 320 G142R or S143L

HE42 IgG1 2560 N145K or N158D mouse serum 640 nd

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Legend to figures 660

Fig. 1 Amino acid differences between the MN/10 and BJ/92 HAs. Shown is the 661

alignment of residues 1-320 of the mature HA1 sequence. Other seasonal H3N2 viruses 662

included are: A/Wuhan/359/1995 (WH/95), A/Panama/2007/1999 (PA/99), 663

A/Wisconsin/67/2005 (WI/05) and A/Texas/50/2012 (TX/12). Dots denote residues that 664

are identical to those in MN/10 HA. Residues in antigenic sites A and B are shaded in 665

green and magenta, respectively. 666

667

Fig. 2 The location of HA amino acids that define the antigenic difference between 668

A(H3N2)v and the ancestral seasonal H3N2 viruses. The monomers in HA trimer of 669

A/Hong Kong/1/1968 (PDB: 4FNK) are shown in light gray, cyan and dark gray. The 5 670

key residues that define the antigenic difference between MN/10 and BJ/92 are 671

highlighted in green. Antigenic site B residue 155 is labeled blue, and the 4 antigenic site 672

A residues tested are in magenta. Residues Y98, S136, W153, T155, H183, G186, D190, 673

L194, L226 and S228 are colored yellow to depict the location of the RBS. The image 674

was generated with PyMol software (Delano Scientific). 675

676

Fig. 3 Minimum amino acid changes in HA that switched the antigenic phenotype of 677

MN/10 and BJ/92. Reassortant MN/10 virus with residues at positions 156, 158, 189 and 678

193 mutated to those present in BJ/92 (156/158/189/193) has an antigenic phenotype 679

typical of wt BJ/92, while reassortant BJ/92 virus with residues 156, 157, 158, 189 and 680

193 mutated to those in MN/10 (BJ156/157/189/193) exhibited an antigenic phenotype 681

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similar to wt MN/10 virus. Each bar represents the average of the Log2 values of HI titers 682

obtained from two independent assays. The dotted line indicates the detection limit. 683

684

Fig. 4 Correlation between HI titers of ferret MN/10 antiserum and binding to (A) SiaTn 685

and (B) 6-SiaTF sialylglycopolymers. Data are plotted as HI titers of ferret antiserum 686

raised against MN/10 virus versus receptor binding affinity (1/Kd, 1/µM of Neu5Ac) of 687

wt MN/10, wt BJ/92 and 5 mutants, 156/158/189/193, 155/156/158/189/193, 688

156/157/158/189/193, BJ156/158/189/193, and BJ156/157/158/189/193. Means are 689

represented as dots and linear regression is represented by dashed line. 690

691

Fig. 5 Binding of MN/10 and BJ/92 viruses by a panel of HA-specific MAbs. The 692

binding was measured by ELISA with plates coated with indicated wt and mutant viruses, 693

which was detected by each MAb (1 µg/ml) and peroxidse-conjugated anti-mouse IgG. 694

Serum from the mouse immunized for MAb generation, which has an HI titer of 640 695

against wt MN/10, was included as a positive control. The OD490 values generated with 696

mutant viruses and wt BJ/92 were normalized to those of wt MN/10 virus. Shown are the 697

averages of 2 independent assays run in duplicate wells, and SDs are indicated by error 698

bars. 699

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