JGV Papers in Press. Published July 30, 2014 as doi:10.1099/vir.0.067819-0

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Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1)

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influenza virus hemagglutinins using monoclonal antibodies and escape mutants

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Miguel Retamal, Yacine Abed, Jacques Corbeil and Guy Boivin* Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, Québec, Canada

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Running title

: HA escape mutants in A (H1N1) viruses.

Contents category: Animal, RNA viruses

Abstract count: 244 Text count: 4932 Inserts: 2 figures, 6 tables

*Corresponding author:

Guy Boivin, MD

CHUL, room RC-709 2705 blvd Laurier, Sainte-Foy, Québec, Canada G1V 4G2 Tel : (418) 654-2705 Fax : (418) 654-2715 E-mail : [email protected]

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1

SUMMARY

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Monoclonal antibodies (MAbs) constitute an important biological tool for influenza virus hemagglutinin (HA)

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epitope mapping through generation of escape mutants, which could provide insights into immune evasion

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mechanisms and may benefit the future development of vaccines. Several influenza A(H1N1) pandemic 2009

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(pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous

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seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Herein, we produced MAbs

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against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated

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using MAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the

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pdm09 HA protein revealed 11 escape mutations including 3 that were previously described (G172E, N173D

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and K256E) and 8 novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T, G478E). Among the 6

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HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), 3 (G172E, N173D and

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K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I,

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K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic

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sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D

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mutation (Sb site), present in 3 different Bris07 escape mutants, appear as an immunodominant epitope in

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that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of

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further characterizing the HA of additional H1N1 strains.

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1

INTRODUCTION

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Influenza viruses possess two major surface glycoproteins, the hemagglutinin (HA) and the neuraminidase

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(NA), that are exposed to the host immune system. These proteins determine the antigenic specificities of

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influenza A viruses allowing their classification into 18 HA (H1-H18) and 11 NA (N1-N11) subtypes (Wu et al.,

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2014). Influenza A viruses of the H1N1 subtype, together with H2N2 and H3N2 viruses, have been present in

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human epidemics and pandemics over the last century. An avian-origin influenza A(H1N1) virus caused the

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most severe pandemic in 1918 that resulted into >40 million deaths (Reid et al., 2000). Oseltamivir-resistant

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influenza A/Brisbane/59/07-like (H1N1) viruses have also been associated with major seasonal influenza

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epidemics between 2007 and 2009 (Holder et al., 2011). In April of 2009, a swine-origin influenza

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A(H1N1)pdm2009 strain emerged causing the first influenza pandemic of the 21 st century (CDC, 2009; Cutler

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et al., 2009; Fraser et al., 2009).

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Monoclonal antibodies (MAbs) constitute an important biological tool for epitope mapping through the

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generation of escape mutants (Caton et al., 1982; Gerhard et al., 1981; Kohler & Milstein, 1975; Lubeck &

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Gerhard, 1981; Webster et al., 1982). The characterization of the influenza A HA protein was initially

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performed using the influenza A/PR/8/1934 (H1N1) reference strain (Caton et al., 1982). In that report, four

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major antigenic sites were identified. These included the ‘specific’ Sa and Sb sites and the ‘cross-reactive’ Ca

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and Cb ones. In addition, as Ca was not a contiguous polypeptide in the primary protein structure, it was split

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into Ca1 and Ca2 regions, which form hypervariable domains into the tertiary globular protein structure. The

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antigenic sites described for influenza A/PR/8/1934 HA have served as a reference model for characterizing

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the antigenicity of different H1N1 strains although considerable antigenic differences, due in large part to their

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swine-, avian-, or human-origins, exist between them. (Gerhard et al., 1981; Lubeck & Gerhard, 1981;

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Robertson & Engelhardt, 2010). More recently, the antigenicity of the A(H1N1)pdm09 virus has been reported 3

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(Hancock et al., 2009; Steitz et al., 2010). Of note, most of the early influenza escape mutant studies were

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carried out using hen egg allantoic fluid grown viruses as a source of antigen (Laver & Webster, 1976; Wood

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& Williams, 1998). However, host-adaptation mutations have frequently been reported when virus is egg-

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adapted (Robertson et al., 1995; Robertson et al., 1993). Moreover, post-translational modifications, mainly

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glycosylation and disulfide bridging, are different in mammalian eukaryotic systems as compared to egg-,

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bacteria-, yeast- or plant- derived antigens. These post-translational modifications of the viral antigen may lead

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to alteration of phenotypic and potentially immunogenic properties of the HA protein (Hart, 1997; Hausmann et

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al., 1997).

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In this study, we created a panel of MAbs against the HA protein of A/California/04/2009 (pdm09) and

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A/Brisbane/59/2007 (Bris07) viruses expressed in human cells. We then mapped the escape mutant epitopes,

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which provide valuable information not only on eventual drifts but for vaccine improvement as well.

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RESULTS

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Comparison of HA antigenic sites demonstrates a significant shift in 2009. The HA antigenic sites from

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selected H1N1 viruses including A/Quebec/144147/2009, A/California/04/2009, A/South Carolina/1/1918,

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A/PR/8/1934 and A/Brisbane/59/2007 were compared (Table 1). There was a strong a.a. identity (98%)

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between the A/Quebec/144147/2009 and A/California/04/2009 proteins, which differ by only one residue

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(T/S220) part of the Ca1 antigenic site. A relatively high a.a. identity (75%) was also seen between

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A/Quebec/144147/2009 and A/South Carolina/1/1918 HA antigenic sites. By contrast, a low a.a. identity (15%)

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was observed between A/Quebec/144147/2009 and A/Brisbane/59/2007 HA antigenic sites. In fact, all 13 a.a.

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(residues 200-212) constituting the Sb antigenic site and 8 a.a. (residues 154-159, 238 and 239) constituting

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the Ca2 antigenic site were completely different between these two strains. In addition, A/PR/8/1934 exhibited

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identities of 23% and 53% with A/Quebec/144147/2009 and A/Brisbane/59/2007 antigenic sites, respectively

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(Table 1).

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Immunoenzymatic properties of monoclonal antibodies reveal differential specificities towards H1-

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pdm09 and H1-Bris07. Screening of hybridomas using enzyme-linked immunosorbent assay (ELISA),

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hemagglutinin inhibition assay (HAI) and Western blot analysis, enabled us to select a total of 33 MAbs (Table

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2). These included 20 MAbs obtained from the mice immunized with the pdm09 antigens (H1-pdm09 MAbs)

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and 13 MAbs obtained with the Bris07 antigens (H1-Bris07 MAbs). In the homologous ELISA experiments, all

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H1-pdm09 MAbs and H1-Bris07 MAbs were positive when tested against their respective virus antigen. In the

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heterologous ELISA experiments testing H1-pdm09 MAbs vs Bris07 antigen and H1-Bris07 MAbs vs pdm09

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antigen, 15 H1-pdm09 MAbs were specific for pdm09, 5 H1-Bris07 MAbs were specific for Bris07, and the

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remaining 13 MAbs (5 H1-pdm09 and 8 H1-Bris07) were cross-reactive.

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Generation of escape mutants from MAbs expressing high binding affinity. After 4 passages in

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MDCKα2-6 cells of either wild-type pdm09 or Bris07 viruses grown in presence of high concentrations of a

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single MAb, a total of 24 escape mutants (17 for pdm09 and 7 for Bris07) were generated (Table 3). The

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isotype and binding affinity were further determined for MAbs that induced these escape mutants. Ten IgG, 3

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IgM and 1 IgA isotypes were identified among H1-pdm09 MAbs, in addition 5 IgG and 3 IgM isotypes were

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identified among H1-Bris07 MAbs. The KD values of purified MAbs as determined with the SKI Pro instrument

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ranged between 1.4 and 47.0 nM for H1-pdm09 MAbs and between 0.2 and 44.3 nM for H1-Bris07 MAbs.

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Seven representative escape mutants from pdm09 virus and 6 from Bris07 virus were tested along with their

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related WT virus in a microneutralization (MN) assay using their respective MAb (Table 4, A and B). All tested

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escape mutants but one (V3P6) were not neutralized by their corresponding MAb, whereas the WT virus was

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neutralized at titers ranging from 1/16 to >1/256. Escape mutants from pdm09 and Bris07 viruses along with

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WT viruses were also tested by ELISA using the respective selecting MAb (Figure 1). The related histogram

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demonstrated a general decrease of MAb recognition in presence of the respective escape mutants.

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Nonetheless they were not affected to the same extent. Apart from the control V1A5 expected to react equally

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with both mock-escape and wild-type, we also notice that V14D11(2) could still recognize its corresponding

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MAb. Similarly, V3E6 and V7B2 still reacted with their MAb.

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HA protein sequence analysis of escape mutants revealed single or double mutations inside and

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outside predicted antigenic sites of A/PR/8/1934. Sequencing of the HA gene revealed that 15 pdm09

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escape mutant variants obtained with our MAbs contained a single HA amino acid (a.a) substitution, whereas

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the other 2 variants had 2 HA mutations (Table 5). Of note, no HA mutations were found in viruses not

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subjected to MAb pressure. The T89R mutation, within the Cb antigenic site, was found in V2F8, V7B6 and

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V14D11 variants. The F128L mutation (between Cb and Sa antigenic sites) was identified in variants V5A2

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and V14B1, whereas the K256E mutation (2 a.a. from Ca1 antigenic site) was identified in V2H2, V3E6 and 6

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V8B10 variants. Escape mutants bearing mutations G157E (Ca2 antigenic site), G172E/ N173D/ K180E (Sa

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antigenic site), A212E (Sb antigenic site), R269K (15 a.a. from the antigenic site Ca1) and G478E (within HA2)

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were isolated once with different MAbs. In total, six of the 11 (54%) mutations were located within known

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antigenic sites, including 3 of 6 (50%) in the Sa antigenic site. The N311T escape mutation was obtained

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alone in V4E3 and V9G6 variants, and as an accompanying mutation in V2F12 and V14D11(2) variants, as

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well as with highly diluted (1/10,000) anti-pdm09 ferret hyperimmune sera (FHS) (Abed et al., 2011).

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Sequencing of the HA gene revealed the presence of 7 Bris07 escape mutant variants (Table 5). Three MAbs

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(3P6, 6B3 and 6B5) induced the same N203D mutation (Sb antigenic site), either alone as in V3P6 or

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accompanied by an additional A231T mutation as for V6B3 and a H54N substitution in V6B5. With the

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exception of the N203D mutation (Sb antigenic site), all Bris07 escape mutations were found outside of

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predicted antigenic sites. For both pandemic and seasonal escape variants, the NA gene was sequenced to

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identify possible mutations that contributed to the new phenotypes. However, we did not find a NA mutation in

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any of the escape mutant variants listed in Table 5, except for V3D11 which harboured the T413P mutation in

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the NA gene.

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Escape mutations are found on the surface of the HA1 subunit protein. The antigenic sites are located on

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the HA1 subunit of the HA protein. The vast majority of our escape mutations from Table 5, except one for

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pdm09 variants (G478E) and one for Bris07 variants (K464E), were located within the HA1 protein subunit.

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On Figure 2, escape mutations reported in Table 5 were mapped onto the tertiary structure of the HA1 protein

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subunit. The 10 escape mutations identified among the pdm09 variants (A), and 6 escape mutations identified

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among the Bris07 variants (B), are shown. These mutations are clearly exposed to the surface and, if not

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within the antigenic sites, they are in close proximity to them (C).

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Prevalence of escape mutations in circulating viral strains. The frequency of HA escape mutations found

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in the NCBI database using the Basic local alignment search tool (BLAST) is reported in Table 6. For pdm09

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viruses, the most frequently found mutations were 157E, 172E, 173D and 180E which were detected between

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15 to >50 times. The 212E mutation was found 6 times in human strains and 11 times in swine strains,

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whereas all other mutations were found in 5 strains or less. Two of them, 89R and 311T were found only in

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swine-reported isolates and 4 (128L, 212E, 256E, 269K) were reported in both human and swine strains. The

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HA2 mutation 478E was found in 2 human strains only. For Bris07 viruses, three escape mutations (203D,

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231T, 314L) were frequently found in more than 50 seasonal H1N1 strains, whereas all other mutations were

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not found in any other strain. Another BLAST analysis on NCBI database aimed at finding concomitant escape

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mutations did not identify more than two mutations on any strain. Only A/Moscow/01/2009(H1N1) contained

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two escape mutations (172E and 173D). For the seasonal Bris07 strain, no concomitant mutations were found

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in any strain.

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1

DISCUSSION

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Characterizing the influenza HA antigenic sites may provide important information related to influenza drifts

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and vaccine efficacy. For such purposes, escape mutants induced by monoclonal antibodies could serve as

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valuable biological tools. In this study, we intended to generate a panel of MAbs and establish an epitope

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map for recent pandemic and seasonal influenza H1N1 viruses using the escape mutants approach. Our

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analysis of the HA antigenic sites from different pandemic and seasonal influenza H1N1 viruses highlighted

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the magnitude of the 2009 pandemic shift (Table 1). We also confirmed the identity gap of both viruses in term

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of antigenic sites with the reference strain A/PR/8/1934.

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Most neutralizing antibodies elicited during influenza infection or vaccination target immunodominant epitopes

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on the globular head region of the HA protein, which leads to specific strain protection (Benjamin et al., 2014).

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In our study we focused on MAbs that demonstrated a significant HAI and neutralizing activity against Bris07

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and pdm09 HA proteins. Despite the fact that all MAbs tested positive in homologous ELISA experiments

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(Table 2), a few of them exhibited negative results in either HAI assay or Western blot, which could be

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explained by specific and non-specific biochemical properties such as their heavy chain composition (isotype),

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glycosylation status, and the regions to which they are directed. The fact that the pdm09 HA was able to

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generate a higher number of specific MAbs than Bris07, tends to support the idea that the new epitopes

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brought by this virus during the 2009 pandemic are more immunogenic than the old epitopes already present

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on the seasonal virus.

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The ability of neutralizing MAbs to select for escape mutants that harbour punctual mutations on the antigenic

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sites has been previously reported (Caton et al., 1982; Lubeck & Gerhard, 1981; Webster et al., 1982). In this

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study, we sought to determine if all neutralizing MAbs could induce the emergence of escape mutants. Our 9

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results from Table 2 and 3 show that only 14 among the 20 pdm09 and 6 among the 13 Bris07 neutralizing

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MAbs induced escape mutants in at least 3 experimental attempts. In order to assess the influence of the

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binding affinity on the emergence of escape mutants, we used the SKI Pro interferometry system for the

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determination of KD values. The MAbs that were associated with the emergence of escape mutants

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demonstrated a relatively high-affinity as the KD values were found to be within the nanomolar (nM) range

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(Malarchuk & Irvin, 2010). However, we failed to generate escape mutants using 1B6 (an IgG1) and 7B7 (an

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IgM) MAbs despite they also had a binding of high affinity. Thus, it appears that neutralizing activity and high-

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binding affinity are probably not the only factors involved in the in-vitro selection of escape mutants. For

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instance, as frequently reported, the high error mutation rate in the polymerase of RNA viruses is probably a

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significant factor determining the existence or appearance of the escape mutants during this type of in vitro

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experiments (Domingo & Holland, 1997). A reason for which not all high-affinity neutralizing MAbs were able

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to generate escape mutants could be explained by the fact that some mutations are too functionally important.

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This type of mutations would generate defective particles that will never outgrow the wild-type fully functional

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virus. Testing of the escape mutant viruses versus the wild-type pdm09 or Bris07 viruses against the MAbs as

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shown in Table 4 (MN) and Figure 1 (ELISA) gave similar results. Indeed, much lower results in neutralization-

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inhibition (MN) and OD450 values (ELISA) were obtained for the escape mutant viruses than for their respective

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wild-type viruses. However, the lack of antibody recognition was more obvious using the MN than the ELISA

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assays. The latter is probably due to the similarity between the MN assay and the protocol used to generate

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escape mutants, which basically relies on neutralization-inhibition as the selective parameter.

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As shown in Table 5, of the 11 escape mutations identified within the HA protein (HA1+HA2) in our pdm09

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background, 6 were located in the A/PR/8/1934 (H1N1)-based predicted antigenic sites (Brownlee & Fodor,

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2001; Caton et al., 1982; Lubeck & Gerhard, 1981). Importantly, 8 new escape mutations (T89R, F128L,

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G157E, K180E, A212E, R269K, N311T and G478E) were generated for the first time to our knowledge. Four 10

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of these newly-identified mutations (T89R, G157E, K180E, A212E) are part of recognized antigenic sites i.e.,

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Cb, Ca2, Sa and Sb, respectively. The G172E mutation has been previously reported in several studies (Chen

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et al., 2014; O'Donnell et al., 2012; Rudneva et al., 2012) whereas the mutation N173D has been reported by

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a single group (Rudneva et al., 2012). These two mutations and our newly found K180E mutation are part of

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the Sa antigenic site. The latter site gave rise to 3 different escape mutants, suggesting that this site offered

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higher immunogenic diversity within the immunized mice. We could anticipate that a small molecule distortion

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could expose to the immune system residues at the extremity of an antigenic site such as for escape mutation

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K256E (2 a.a. from Ca1 site); however, it is harder to anticipate an epitope exposition of F128L (16 a.a. from

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Cb site and 13 a.a. from Sa site) or R269K (14 a.a. from Ca1 site). The complete HA protein of H1N1 viruses

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is 566 a.a. long, but is later cleaved into the HA1 subunit of 338 a.a. long and the HA2 subunit of 228 a.a.

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long. Thus, the HA2 mutation found with MAb 2F8 (G478E- prior cleavage), is located at a.a.140 of the HA2.

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This site is located in the HA2 stem farther than the reported conserved sites (K51, D109 and D112)

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responsible for membrane fusion (Thoennes et al., 2008).

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The low number of MAbs generated against the seasonal Bris07 virus may constitute a limitation of our study.

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According to Table 5, we obtained 8 different mutations at 7 different residues in our Bris07 escape mutants.

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Only one escape mutation (N203D) was located within antigenic site Sb for 3 of the escape variant viruses

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(V3P6, V6B3 and V6B5). Notably, to our knowledge, all 8 escape mutations (H54N, N55D, N55K, L60H,

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N203D, A231T, V314I and K464E) in the Bris07 background have not been reported. Unlike the Sa antigenic

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site in pdm09 background variants, the Sb site in Bris07 background variants does not seem to show

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immunogenic diversity, but rather a single immunodominant amino acid (N203D) that was targeted by 3

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different hybridomas. In our study, we did not find mutations on the same residue for both pdm09 and Bris07

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HA; however, we generated an escape mutation N203D for the Bris07 HA and another group of investigators

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generated similar and nearby escape mutations (A203T and D204E) from their local pdm09 virus (Yasugi et 11

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al., 2013). Thus, the 203 position appears as a potential target for neutralizing both seasonal and pandemic

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viruses simultaneously. The Bris07 mutation on HA2 (K464E-prior cleavage) selected with MAb 3D11, is

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located at position 126 of the HA2 protein after cleavage. This position is also located on the HA2 stem

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subunit, farther than the conserved sites responsible for fusion with host cell membrane (Thoennes et al.,

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2008). However, this a.a. is located closer to these important sites than the HA2 mutation (G478E) found in

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pdm09. Interestingly, the escape mutant V3D11 was also the only virus with a mutation in the NA gene

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(T413P), which may compensate for an unfavorable HA2 mutation. Moreover, this MAb (3D11) also showed

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the highest affinity of all neutralizing MAbs with a low K D value of 0.2 nM (Table 3).

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Usually, escape mutation experiments performed with MAbs generate 1, 2 or 3 amino acid changes. These

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mutations occur most commonly at the direct site of attachment of the MAb, but they may also consist of

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mutations due to host adaptation (Robertson et al., 1993). However, in such situation, these variants would

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not affect ELISA test results since the epitope would not be lost. Another possibility is the emergence of

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compensatory mutations that occur in an attempt to keep viral fitness (Abed et al., 2014). The fact that N311T

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was the only mutation reported when diluted hyperimmune sera was used and that 5 MAb variants contained

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this mutation, suggests that it might be a strategic mutation allowing the HA to take a very different antigenic

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conformation and to evade MAb pressure by revealing new epitopes.

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Although the primary a.a. sequences on Table 5 showed mutations at 3 very different regions of the HA1

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molecule i.e., the start of the protein region (a.a. 54 to 60), the antigenic sites region (a.a. 87 to 254), and the

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far end of the protein region (beyond a.a. 300), the 3-D positioning of those mutations highlights mainly two

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regions as shown on Figure 2. One region comprised a small cluster of mutations, including not only H54N,

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N55D, N55K and L60H mutations, but also N311T and V314I mutations as well, which represent the start and 12

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the end of the protein stem. The other region comprised a large cluster of mutations representing the globular

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head encompassing previously-reported antigenic sites and all other HA1 mutations. Moreover, such epitope

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mapping localized mutations inside and outside of the antigenic sites at positions that are accessible to

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antibodies. Our BLAST analysis provided valuable information on eventual drifts. It suggests, for instance, that

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substitutions T89R and N311T in the pdm09 background, so far reported only in swine viruses, could be

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eventually transferred to humans highlighting the need for constant surveillance efforts.

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To summarize, we obtained a panel of MAbs against the pdm09 and Bris07 HA proteins, using mammalian

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cells for both antigen production and escape mutants generation. Forty five percent of the pdm09 escape

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mutations and 87.5% of the Bris07 escape mutations were located outside the reference antigenic sites of

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A/PR/8/1934 (H1N1). Thus, due to the heterogeneity of H1N1 HA proteins, additional strains should be

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antigenically characterized in order to refine and update the epitopes defining the antigenic sites. Such

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information will help in the design of cross-reactive vaccines. Our results could also be used to improve bio-

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mathematical models aimed at predicting strain variability. (Deem & Pan, 2009; Lee & Chen, 2004).

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1

METHODS

2 3

Mouse immunizations and hybridomas

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BALB/c mice were immunized sub-cutaneously (s.c.) using Freund incomplete adjuvant (FIA) (Thermo Fisher,

5

Rockford, IL) and 30 µg of a whole tween-ether inactivated influenza A/QC/144147/2009 preparation (an

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A/California/04/2009-like pdm09 virus) propagated in Madin-Darby canine kidney cells overexpressing ST6-

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Gal I receptor (MDCK α2-6) (Hatakeyama et al., 2005) and purified by sucrose gradients (Arora et al., 1985;

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Stahl-Hennig et al., 1992). Three weeks later, a s.c. boost of FIA with 10 µg of recombinant

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A/California/04/2009 HA protein (recHA) produced in HEK293 human cells (Sino Biologicals, Beijing, China)

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was performed. The boost was repeated at 15-day intervals until serum samples show adequate levels of

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immunization as assessed by ELISA testing (see below). Another group of mice was immunized as per the

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previous protocol but with an A/Brisbane/59/2007-like isolate and then with the recombinant Bris07 HA

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produced in human HEK293 cells (Sino Biologicals). In both groups, one final pre-fusion boost was done

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intravenously with 10 µg of the respective recHA in PBS, 3 days prior to the hybridoma-generating cellular

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fusion. The cellular fusions between myeloma cells P3X63Ag8 (ATCC, Manassas, VA) and splenocytes were

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carried out with the ClonaCell™-HY Hybridoma Kit (Stemcell Technologies, BC, Canada) following the

17

manufacturer’s instructions. Once identified as positive by ELISA and HAI (as per sections below), the

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hybridomas were cloned to approximately 5 cells per well in 96-well round bottom plates (Corning, Tewksbury,

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MA) using Hybridoma Medium (Stemcell Technologies).

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Immunoenzymatic screenings

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Hybridomas were screened with ELISA using Maxisorp 96-well plates (Thermo Fisher Scientific, Waltham,

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MA) that were coated overnight with 50 µL of 10 to 50 ng of either pdm09 or Bris07 whole-inactivated viruses

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into carbonate buffer (pH 9.6). Plates were washed 3 times with PBS-Tween 0.05% (PBS-T) and saturated 14

1

with 5% BSA for 3 h at room temperature (RT). Plates were washed again 5 times with PBS-T and 50 µL of

2

hybridoma culture supernatant were then added to each well for 1 h at RT. After seven washes with PBS-T, 50

3

µL of a 1/2500 dilution of anti-mouse IgG HRP H+L chains (Promega, Madison, WI) were added to the wells

4

for 1 h. Plates were washed 7 times with PBS-T and then 50 µL of TMB substrate (Fitzgerald Laboratories,

5

Acton, MA) were added for 10 min and the reaction was stopped by adding 50 µL of H 2SO4 2N solution. The

6

OD450 was read with a spectrophotometer for 0.1 sec. Putative positive wells containing the desired hybridoma

7

were identified as wells having both an OD450 > 0.2 and > 3 times that of the negative control value.

8

Hybridomas that tested positive against the whole virus and the recHA by ELISA were subsequently analyzed

9

by HAI (WHO, 2002). For ELISA testing on escape mutants and their wild-type counterparts, the same

10

protocol was used except that plates were coated in triplicate with 32 HA units of viral antigen as the first step.

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Western blotting

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Five µg of sucrose-gradient purified virus proteins from A/Quebec/144147/2009 or A/Brisbane/59/2007 were

14

separated into a 12% SDS-PAGE (Arora et al., 1985; Laemmli, 1970). The SDS-PAGE was transferred on a

15

nitrocellulose membrane (Towbin et al., 1992). Strips of nitrocellulose were saturated with 7% milk in tris

16

buffered saline 0.1 % Tween-20 (TBS-T) for 2 h at RT. Strips were washed 3 times with TBS-T and incubated

17

with 5 mL of hybridoma culture supernatant in TBS-T 0.2% milk at a ratio of 1:1. After 2 washes of the strips

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with TBS-T, 5 mL of TBS-T 0.1% milk with anti-IgG mouse (H+L) HRP conjugated (1/2500 dilution in PBS)

19

were added for 1 h at RT. Strips were washed as mentioned previously and revealed with TMB substrate #

20

W4121 (Promega).

21 22

Microneutralization

23

For MN assay, 50% tissue culture infectious dose (TCID50) viral titers were determined in MDCK α2-6 cells.

24

MAbs were 2-fold serially diluted in PBS with one TCID50 dose of virus. The plates were incubated for 1 h at 15

1

RT to allow for virus antibody interaction. The contents of each well were then transferred onto microtiter

2

plates with confluent monolayers of MDCK α2-6 cells. After 1 h of further incubation at 37°C, the virus/MAbs

3

mixture was removed and washed twice with 150 µL per well of PBS. After the last wash, 200 µL of infection

4

medium (DMEM High-Glucose Ref# 11995; Life Technologies, Grand Island, NY) containing 2 µg/mL L-1-

5

tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, 0.1% BSA and 1x HEPES and

6

antibiotics were added. The plates were further incubated at 37°C in 5% CO 2 and cytopathic effects (CPE)

7

were monitored for 7 days post-inoculation. The MN titer was defined as the inverse of the serum dilution

8

immediately preceding the wells exhibiting CPE (Skowronski et al., 2014).

9 10

Evaluation of binding affinities by KD determination

11

The binding affinity of the different MAbs was evaluated with optical interferometry using the SKI Pro

12

instrument (Silicon Kinetics, San Diego, CA). The isotype of the MAbs was first determined with an isotyping

13

kit (Rockland, Gilbertsville, PA). Then, MAbs were purified with AcroSep Protein A HyperDF-1mL columns

14

(PALL Life Science, Ann Arbour, MI). The MAbs were then dialyzed on nanosep 10K OMEGA columns (PALL

15

Life Science) using PBS as matrix buffer. As a first step, 5 µg of recombinant HA were immobilized on the

16

sample channel and 5 µg of BSA were immobilized on the reference channel of a carboxychip (Silicon

17

Kinetics). Immobilization took place at 6 µL/min for 7 min followed by quenching with ethanolamine 1M pH8.5

18

(Silicon Kinetics) and re-equilibration in H2O. The chip was then equilibrated with PBS for 10 min and 90 µL of

19

MAbs diluted in PBS at 0.1mg/mL were passed through both sample and reference channels at 10 µL/min

20

and this was followed by a 6 h dissociation period. The optical displacement difference (OPD) in nm between

21

both channels allowed for the establishment of a binding curve followed by a dissociation curve. The SKI Pro

22

software algorithm was used to calculate Kon, Koff and Kobs values, from which a KD value was estimated

23

(Latterich & Corbeil, 2008; Sanchez et al., 2010; Stephan et al., 2014).

24 16

1

Generation of escape mutants and epitope mapping

2

The wild-type pdm09 and Bris07 viruses were immunocomplexed with different MAbs by mixing 50 µL of 2-

3

fold serial dilutions of virus in PBS with 50 µL of undiluted hybridoma supernatant for 30 min at RT. Ninety-six-

4

well plates containing MDCK α2-6 cells were washed twice with PBS and adsorbed with the

5

immunocomplexed viruses at 37oC / 5% CO2 for 30 min. Three washes with 150 µL of PBS per well were

6

done to remove unadsorbed viruses, then 200 µL of infection medium (see above) were added to each well

7

followed by an incubation of 3 to 7 days at 37oC/ 5% CO2. Wells corresponding to the highest viral dilution

8

giving CPE were selected. These steps of escape mutant selection were repeated twice in order to enrich the

9

escape mutant viral population. At the 4th and last passage, infection was performed in a 6-well plate and 3 to

10

5 aliquots of 1 mL containing escape viral mutants were frozen at -80 oC. Aliquots of 150 µL of infected cell

11

culture supernatant were extracted using an RNA extraction kit (Qiagen, Hilden, Germany) followed by a

12

reverse-transcription step with the Superscript II (Life Technologies). The PCR was performed with Taq DNA

13

polymerase (Life Technologies) and HA/NA specific primers (available upon request). PCR products of the HA

14

and NA genes were sequenced using the Applied Biosystems instrument ABI3730xl. The sequences were

15

aligned using CLC Sequence viewer (v.6.6.2, Prismet, Denmark). The mutations were mapped onto the 3-D

16

structure of the HA1 using Protein Data Bank File #3AL4 available on the RCSB-PDB website

17

(http://www.rcsb.org/pdb/home/home.do). Finally, the PyMOL Molecular Graphics System (Schrödinger,

18

Portland, OR) was used to highlight the 3-D location of the main HA mutations identified in this study (Yiu &

19

Chen, 2014).

20 21

Mutation prevalence and BLAST analysis

22

The identified escape mutations were inserted in the middle of a 10-a.a. sequence stretch of either pdm09 or

23

Bris07 HA proteins. The mutated sequences were then searched using the protein Basic Local Alignment

24

Search Tool in the National Center for Biotechnology Information (NCBI) database. Complete H1 proteins from 17

1

pdm09 and Bris07 viruses containing all escape mutations identified in this study were also BLAST analyzed

2

for identification of similar reported strains.

3 4 5

ACKNOWLEDGEMENTS

6

This study was supported by the Canadian Institutes of Health Research and GlaxoSmithKline Canada (grants

7

230187 and 229733 to GB).

8

MR has received a PCIRN (PHAC/CHIR Influenza Research Network) scholarship for his PhD studies.

9 10 11 12 13 14

18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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21

1 2

Table 1. Comparison of antigenic sites (amino acid sequences) of H1 influenza viruses. Sa

141 P P P P P

142 N N N N N

170 K K K K K

171 K E N K K

172 G G G G G

173 S S L N N

174 S Y Y S S

176 P P N P P

177 K K L K K

Sb

200 P N N S S

201 T S I T T

202 G K G S S

203 T E N A A

204 D Q Q D D

205 Q Q K Q Q

206 Q N A Q Q

207 S L L S S

208 L Y Y L L

A/South Carolina/1/1918 A/PR/8/1934 A/Brisbane/59/2007 A/California/04/2009 A/Québec/144147/2009

178 L L S L L

179 S K K S S

180 K N S K K

181 S S Y S S

211 N E E N N

212 A N N A A

3 A/South Carolina/1/1918 A/PR/8/1934 A/Brisbane/59/2007 A/California/04/2009 A/Québec/144147/2009

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Ca1

A/South Carolina/1/1918 A/PR/8/1934 A/Brisbane/59/2007 A/California/04/2009 A/Québec/144147/2009

Ca 2

A/South/Carolina/1/1918 A/PR/8/1934 A/Brisbane/59/2007 A/California/04/2009 A/Québec/144147/2009

Cb

184 N K N N N 154 S H H P P

A/South Carolina/1/1918 A/PR/8/1934 A/Brisbane/59/2007 A/California/04/2009 A/Québec/144147/2009 H1N1 Influenza Strains

26 27

183 V N N I I

185 N K K D D 155 Y E N H H

86 L P L S S

Nb of a.a Identical to A/Quebec/144147/2009

87 L L L L L

186 K G E K K 156 A G G A A 88 L L I S S

187 G K K G G

220 S S S S T

157 G K E G G

158 A S S A A

89 T P S T T

90 A V K A A

221 S N H S S 159 S S S K K 91 S R E S S

209 Y Q H Y Y

222 K Y Y R R

252 E P P E E

238 R D D R R 92 S S S S S

TOTAL antigenic sites Nb of a.a % Identity* Identical to vs A/PR/8/1934 A/Quebec/144147/2009 A/South Carolina/1/1918 40 75 16 A/PR/8/1934 12 23 53 A/Brisbane/59/2007 8 15 28 A/California/04/2009 52 98 14 A/Québec/144147/2009 53 Reference (100%) 12 *%Identity = (Nb of a.a identical to reference strain / Total Nb of a.a in antigenic sites) x 100%. Shaded a.a have been selected by MAbs pressure as part of this study.

210 Q N T Q Q 253 P G G P P

254 G D D G G

239 D Q Q D D 93 W W W W W TOTAL antigenic sites % Identity* vs A/PR/8/1934 30 Reference (100%) 53 26 23

22

1 2 3 4 5

Table 2. Immunoenzymatic properties and cross-reactivities of monoclonal antibodies directed against the hemagglutinin of the 2009 pandemic (pdm09) and 2007 seasonal (Bris07) influenza A/H1N1 viruses. Immunoenzymatic characteristics as assessed by:

H1-pdm09 MAbs

HAI

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

+ + + + + + + + + + + + + + + + + +

1B4 2D2 2F8 2F12 2H2 3E6 4E3 5A2 7B6 7F1 8B10 9B4 9D10 9G6 11E9 12B12 13C1 14A7 14B1 14D11

H1-Bris07 MAbs 1 2 3 4 5 6 7 8 9 10 11 12 13

HAI

*

+ + + + + + + + + + + + +

1B6 3D4 3D8 3D11 3P6 6B3 6B5 6G2 7B2 7B7 8C11 9D4 10F10

*

Western

+ + + + + + + + + + + + + + + + + + + +

Western

+ + + + -

+ + +

†

†

MN

ELISA

ELISA§

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + -

‡

§

pdm09

MN

ELISA

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

‡

§

Bris07

Bris07

ELISA§ pdm09

+ + + + + + + + -

* A positive (+) result is a difference between Ab and negative control of at least 2 antibody dilutions. † A positive (+) result is a naked eye visible band detection at the appropriate MW size. ‡ A positive (+) result is a difference between Ab and negative control of at least 2 antibody dilutions. § A positive (+) result is a OD >3x that of negative control and higher than 0,2000. 450

23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Table 3. Affinity of monoclonal antibodies that selected escape mutant variants from pdm09 and Bris07 A(H1N1) viruses.

H1-pdm09 MAbs

Affinity* K (nM)

Escape Mutant Generated

†

D

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1B4 2F8 2F12 2H2 3E6 4E3 5A2 7B6 7F1 8B10 9G6 14A7 14B1 14D11

Ig M Ig G2a Ig A Ig G1 Ig G2a Ig M Ig G2a Ig G1 Ig G1 Ig G2a Ig M IgG3 IgG1 IgG1

2.1 22.5 6.6 2.4 14.9 9.5 3.8 1.4 47.0 2.4 10.7 26.0 N.D. 9.2

V1B4 V2F8 ; V2F8(2) V2F12 V2H2 V3E6 V4E3 V5A2 V7B6 V7F1 V8B10 V9G6 V14A7 V14B1 V14D11 ; V14D11(2) ; V14D11(3)

1 2 3 4 5 6 7 8

1B6 3D4 3D11 3P6 6B3 6B5 7B2 7B7

IgG1 IgG1 IgM IgG1 IgG1 IgG1 IgM IgM

7.0 21.1 0.2 44.3 23.1 36.0 0.8 1.1

V3D4 V3D11 V3P6 V6B3 V6B5 V7B2 ; V7B2(2) -

H1-Bris07 MAbs

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Isotype

*Affinity in KD is calculated using Kobs, Kon and Koff through SKI Pro system (Silicon Kinetics). † Antigen used to measure affinity is purified recombinant HA from A/California/04/2009 or A/Brisbane/59/2007.

24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Table 4. Microneutralization titers for pdm09 (A) and Bris07 (B) wild-type and escape mutant viruses.

A Monoclonal Antibody

PBS (ctrl) 1B4 2F8 2H2 7F1 7B6 8B10 14A7

B Monoclonal Antibody

PBS (ctrl) 3D4 3D11 3P6 6B3 6B5 7B2

Micro-neutralizations* vs vs A/Quebec/144147/2009 Escape Mutants (wt) Variant WT Virus Titers Virus Titers VPBS V1B4 V2F8 V2H2 V7F1 V7B6 V8B10 V14A7

0 0 0 0 0 0 0 0

WT WT WT WT WT WT WT WT

0 1/16 1/16 1/16 1/16 1/16 1/64 1/64

Micro-neutralisations* vs vs A/Brisbane/59/2007 Escape Mutants (wt) Variant WT Virus Titers Virus Titers VPBS V3D4 V3D11 V3P6 V6B3 V6B5 V7B2

0 0 0 1/16 0 0 0

WT WT WT WT WT WT WT

0 1/32 1/16 1/64 1/16 1/16 >256

*Equivalent quantities of escape-mutant and wild-type virus at one TCID50 were tested with 2-fold dilutions of MAbs, using MDCKα2-6 cells in 96-well plates.

46 47 48 49

25

1 2 3

Table 5. Localization of escape mutations from A/Quebec/144147/2009 and A/Brisbane/59/2007 influenza viruses inside and outside H1 antigenic sites. H1 Hemagglutinin Antigenic Sites*

HA1 Cb

Sa

Ca2

Sa

Sa

Ca1

Ca1

54-60

87-92

128

141-142

154-159

170-174

176-181

183-187

201-212

220-222

231

238-239

252-254

256

269

311

314

464

478

LSTASS

F

PN

PHAGAK

KKGNS

PKLSKS

INDKG

TSADQQSLYQNA

TSR

I

RD

EPG

K

R

N

N

E

G

§

2)

V2F8

N173D

T89R

V2F8(2)

4)

V2F12

5)

V2H2

6)

V3E6

9)

Ca2

HNGKLCK

V1B4

8)

Ca1

H1-Numbering†

1)

7)

Sb

a.a. sequences ‡

MAbs ESCCAPE VARIANTS pdm09

3)

HA2

A212E

V4E3 V5A2

T89R

V7B6

10)

V7F1

11)

V8B10

12)

V9G6

13) V14A7 14)

V14B1

15)

V14D11

T89R

K256E K256E

K256E

N311T

G172E

F128L

R269K N311T

16) V14D11(2)

K180E

17) V14D11(3) VFHS

N311T

F128L G157E

║ ¶

G478E

N311T

N311T

VPBS

V10E5# a.a. sequences ‡

HNGKLCL

MAbs ESCCAPE VARIANTS Bris07§

1)

V3D4

2)

V3D11

3)

V3P6

4)

V6B3

5)

V6B5

6)

V7B2

7) V7B2

4 5 6 7 8

LISKES

F

PN

HNGESS

KNGLY

NLSKSY

KEKEV

IGNQKALYHTEN

SHY

A

DQ

PGD

I

Y

V

V

V314I N203D N203D N203D

H54N N55D N55K/L60H

* Antigenic Sites Cb,Sa,Ca2,

Sa,Ca1 and Sb are as initially described (Caton , 1982). † H1 amino acid numbering of the uncleaved H1 protein starting at first methionine (start codon) (Igarashi , 2010). ‡ a.a. sequences are from A/Quebec/144147/2009 and from A/Brisbane/59/2007 strains. § Variants of viruses A/Quebec/144147/2009 (as pdm09) and A/Brisbane/59/2007 (as Bris07) were obtained by 4 dilution-limitcloning passages of viruses in MDCKα2-6 cells in the presence of high concentrations of MAb. et al.

et al.

K

N

K464E

A231T

║ VFHS is an escape mutant virus obtained with the use of polyclonal antibodies from hyperimmuned anti-pdm09 ferret serum. ¶ VPBS is a wild-type virus grown in the absence of MAb (PBS only), serving as negative control # V10E5 is a wild-type virus grown in the presence of MAb 10E5 that does not recognize pdm09, serving as negative control.

26

Table 6. Prevalence of the encountered escape mutations among circulating influenza strains. BLAST MUTATION*

Frequency

Representative Field Strains† pdm09

1 2

89R 128L

1x in Swine

A/Swine/Minnesota/A0132792/2012 (H1N1)

1x in Human 2x in Swine

A/Cameron/10v-1090/2010 (H1N1) A/Swine/Hong-Kong/3984/1999 (H1N1)

15x Human

A/California/21/2013 (H1N1)

>50x Human

A/Moscow/01/2009 (H1N1)

28x Human

A/California/07/2013 (H1N1)

>50x Human

A/Ancona/310/2009(H1N1)

6x in Human 11x in Swine

A/England/193840010/2009 (H1N1) A/Swine/Minnesota/A076205/2010 (H1N2)

7

157E 172E 173D 180E 212E

8

256E

2x in Human 4x in Swine

A/Rome/676/2009 (H1N1) A/swine/North Carolina/00253/2004 (H1N1)

9

269K

2x in Swine 3x in Human

A/swine/MO/23881/2010 (H1N1) A/Mures/14446/2009 (H1N1)

10

311T 478E

1x in Swine

A/Swine/Nebraska/A01241113/2012 (H1N1)

2x in Human

A/Singapore/KK124/2011 (H1N1)

3 4 5 6

11

A/Finland/630/2009(H1N1)

54N, 55D, 55K & 60H 2 203D 3 231T 4 314I 1

Bris07 0X

NA

>50X Human

A/Boston/22/2009

>50X Human

A/Nigata/08f306/2009

>50X Human

A/mallard/France/710/2002

464E 0X *BLAST analysis were performed using NCBI Standard Protein BLAST algorithm. †Most prevalent example of strain harbouring this mutation is shown. 5

NA

27

1

FIGURE LEGENDS

2 3

Figure 1. ELISA testing of MAbs against wild-type influenza A(H1N1) viruses and their escape mutants.

4

Immunological reactivity of MAbs in Enzyme-Linked Immunosorbent Assay (ELISA) using equivalent

5

quantities of wild-type influenza A/Quebec/144147/2009 or A/Brisbane/59/2007-like viruses or their respective

6

escape mutants as antigen. Triplicates of 32 HA units of virus were coated in 96-well plates and bound with

7

respective MAbs (50 µL hybridoma supernatant). Fifty µL of anti-IgG(H+L) at 1/2500 dilution revealed

8

colorimetric OD450 detectable signals. Three negative controls of ELISA signal were performed: 1) SN

9

Myeloma, which consists of supernatant of myeloma culture, 2) Medium A, which is the cell medium from

10

StemCell Technologies, and 3) 10E5, which is a non-recognizing MAb. Another control providing a positive

11

ELISA signal was also used: 1A5; a MAb which has not selected for escape mutants and which is not

12

neutralizing but recognizes the pdm09 virus. Error bars represent SEM.

13 14

Figure 2. Three-dimensional localization of escape mutations . Sixteen escape mutations have been

15

identified within the HA1 protein sub-unit using our monoclonal antibody panels. (A) Ten mutations obtained

16

from virus A/Quebec/144147/2009 with MAbs directed towards pdm09. (B) Six mutations obtained from

17

Quebec A/Brisbane/59/2007-like isolate with MAbs directed towards Bris07. (C) The five antigenic sites, Ca1,

18

Ca2, Cb, Sa and Sb, of the H1 molecules as previously described on A/PR/8/1934 (Caton

19

shown. The figure was generated using PyMol Molecular Graphic Systems (Schrödinger).

et al.

, 1982) are

28