Broadly Cross-Reactive, Nonneutralizing Antibodies against Influenza B Virus Hemagglutinin Demonstrate Effector Function-Dependent Protection against Lethal Viral Challenge in Mice

While broadly protective antibodies against the influenza A virus hemagglutinin have been well studied, very limited information is available for antibodies that broadly recognize influenza B viruses. Similarly, the development of a universal or broadly protective influenza B virus vaccine lags behind the development of such a vaccine for influenza A virus. More information about epitope location and mechanism of action of broadly protective influenza B virus antibodies is required to inform vaccine development. In addition, protective antibodies could be a useful tool to treat or prevent influenza B virus infection in pediatric cohorts or in a therapeutic setting in immunocompromised individuals in conjugation with existing treatment avenues.

ABSTRACT

Protection from influenza virus infection is canonically associated with antibodies that neutralize the virus by blocking the interaction between the viral hemagglutinin and host cell receptors. However, protection can also be conferred by other mechanisms, including antibody-mediated effector functions. Here, we report the characterization of 22 broadly cross-reactive, nonneutralizing antibodies specific for influenza B virus hemagglutinin. The majority of these antibodies recognized influenza B viruses isolated over the period of 73 years and bind the conserved stalk domain of the hemagglutinin. A proportion of the characterized antibodies protected mice from both morbidity and mortality after challenge with a lethal dose of influenza B virus. Activity in an antibody-dependent cell-mediated cytotoxicity reporter assay correlated strongly with protection, suggesting that Fc-dependent effector function determines protective efficacy. The information regarding mechanism of action and epitope location stemming from our characterization of these antibodies will inform the design of urgently needed vaccines that could induce broad protection against influenza B viruses.

IMPORTANCE While broadly protective antibodies against the influenza A virus hemagglutinin have been well studied, very limited information is available for antibodies that broadly recognize influenza B viruses. Similarly, the development of a universal or broadly protective influenza B virus vaccine lags behind the development of such a vaccine for influenza A virus. More information about epitope location and mechanism of action of broadly protective influenza B virus antibodies is required to inform vaccine development. In addition, protective antibodies could be a useful tool to treat or prevent influenza B virus infection in pediatric cohorts or in a therapeutic setting in immunocompromised individuals in conjugation with existing treatment avenues.

INTRODUCTION

On average, one-quarter of the annual influenza cases are caused by influenza B viruses (1). However, that is not necessarily always the case, as shown by the 2017 to 2018 influenza season in Europe, during which more than 60% of influenza cases were caused by influenza B virus strains (1). Influenza B virus infections are typically less severe than influenza A H3N2 virus infections but more severe than influenza A H1N1 virus infections and are a significant concern in pediatric populations (2). Two lineages of influenza B viruses, the B/Victoria/2/1987-like (Victoria-like, V) and B/Yamagata/16/1988-like (Yamagata-like, Y) lineages, have cocirculated in humans at least since 1983 (3–5). These lineages are defined by the antigenicity and genetic relatedness of the hemagglutinin (HA) surface glycoprotein (Fig. 1A). HA is the major surface glycoprotein of influenza virus and also the main antigen targeted included in influenza virus vaccines. Antibodies that bind to the immunodominant, discrete antigenic sites on the membrane distal globular head domain of the HA are neutralizing in vitro and typically protective in vivo, as they are capable of interfering with the interaction of the HA and sialic acid receptors. However, the globular head domain is highly plastic and undergoes antigenic drift, making it a difficult target for vaccination (6). Broadly protective monoclonal antibodies (MAbs) that target influenza A virus HA have been well characterized, and a large panel of these MAbs has been isolated (7, 8). While a majority of these MAbs have neutralizing activity in vitro, broad protection conferred in vivo by nonneutralizing antibodies via Fc-Fc receptor interactions has been described as well (9–11). Data for broadly protective influenza B virus antibodies are sparse and limited to only a few studies reporting a small number of MAbs capable of displaying broad influenza B virus neutralization capacity in vitro (12–15).

FIG 1.

Phylogenetic tree of influenza B virus HA and immunization strategy. (A) Influenza B virus HA amino acid sequences were aligned and rooted to the ancestral influenza B/Lee/1940 virus HA. The ancestral strains (orange) prior to divergence, antigenically distinct B/Victoria/2/1987-like strains (green), and B/Yamagata/16/1988-like strains (purple) are indicated. Stars indicate recombinant HAs or purified viruses used to test the broad binding capabilities of our MAbs. The scale bar represents a 1% difference in amino acid sequence identity. Sequences were obtained on FluDB or GISAID, and the tree was generated in Clustal Omega and visualized in FigTree. (B) Generation of broadly reactive MAbs against influenza B virus HA. This schematic highlights the strategy used to generate anti-influenza B virus HA MAbs through hybridoma technology. PEG, polyethylene glycol.

Here, we characterized a panel of 22 broadly reactive nonneutralizing antibodies that recognize influenza B virus HA, with the majority of these antibodies binding to the HA stalk domain. A significant proportion of these MAbs protect mice from both morbidity and mortality after influenza B virus challenge. This protection correlates with activity in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay and therefore seems to be driven by Fc-mediated effector functions. Information about the mechanism of protection as well as the epitope specificity of these MAbs will be important for the future design of vaccines that induce broadly protective immunity against influenza B virus infection.

RESULTS

Monoclonal antibody generation.

To generate cross-reactive anti-influenza B virus HA antibodies, female BALB/c mice were immunized with the ancestral B/Lee/1940 (16), B/Victoria/2/87 (V), and B/Yamagata/16/88 (Y) viruses (Fig. 1B). In order to ensure that the antibody response was generated against the HA, rather than the other glycoprotein, neuraminidase (NA) (17), the immunogen from the Yamagata-like lineage was administered as an A/PR/8/34 virus expressing the B/Yamagata/16/88 HA instead of its own (18) as a boost before the fusion. Following hybridoma fusion of the splenocytes from one of these mice to SP2/0 myeloma cells, supernatants from the IgG-producing clones were screened through enzyme-linked immunosorbent assays (ELISAs) for reactivity against baculovirus-expressed, purified influenza B virus HA from B/Lee/40, B/Victoria/2/87, B/Yamagata/16/88, B/Florida/4/06 (Y), and purified B/Florida/4/06 (Y) and B/New Jersey1/12 (V) whole viruses. Cultures secreting polyclonal or nonreactive antibody were discarded, and only IgG isotype antibodies were selected. Overall, 1,133 hybridoma clones were initially picked for screening. Of this, 104 clones passed the screening process against purified viruses and recombinant HAs. Of the 68 IgGs, these 22 MAbs were selected based on high reactivity against the ancestral B/Lee/40 strain while showing the highest cross-reactivity to the other tested strains. The final panel of 22 mouse MAbs was selected for further characterization based on isotype and cross-reactivity as determined by ELISAs.

Isolated MAbs show broad reactivity but no neutralizing activity in vitro.

Next, we assessed the breadth of binding of the antibodies to the diverging lineages of influenza B virus. To do so, ELISAs were carried out against a wide range of purified influenza B viruses spanning approximately 73 years of evolution. Most of the antibodies displayed cross-reactivity to the selected panel of viruses (Fig. 2A). Certain antibodies, like KL-BHA-11C12, displayed a relatively high minimal binding concentration (MBC) to all viruses tested in comparison to the other MAbs in the panel. One MAb, KL-BHA-7F10, showed a lack of binding to all viruses belonging to the Victoria-like lineage and to more recent Yamagata-like lineage viruses, such as B/Florida/4/06 and B/Phuket/3073/13, while binding to all other strains. Another MAb, KL-BHA-7C7, showed a similar lack of binding to recent virus isolates from both lineages. As seen in Fig. 2, all MAbs were capable of binding the older ancestral B/Lee/40 and B/Great Lakes/1739/54 viruses. Sixteen MAbs also showed binding to a recent B/Phuket/3073/13 (Y) virus with a wide range of MBCs, which indicates that the cross-reactivity does not just persist across lineages but also across viruses evolving over time. The absence of binding of several MAbs to B/Phuket/3073/13 can be attributed to the fact that the immunogen selected from the Yamagata-like lineage was B/Yamagata/16/88, an older virus than the counterpart immunogen from the Victoria-like lineage, B/Malaysia/2506/04, which is a more recent isolate. Correspondingly, this might explain the retention in binding to a more recent B/Texas2/13 (V) virus. In summary, our MAb panel includes antibodies that show broad cross-reactivity to influenza B viruses from both lineages, representing a time period of 73 years. The neutralization capability of these antibodies was tested through a plaque reduction neutralization assay against B/Yamagata/16/88 virus, to which all MAbs bind with low MBCs (Fig. 2B). However, all 22 MAbs were incapable of neutralizing B/Yamagata/16/88 and did not result in a reduction in plaque number compared to a positive control.

FIG 2.

Anti-influenza B virus HA MAbs are broadly cross-reactive and nonneutralizing in vitro. (A) Binding profiles of anti-influenza B virus HA MAbs. The isolated MAbs display broad binding profiles for ancestral (orange), B/Victoria/2/1987-like (green), and B/Yamagata/16/1988-like (purple) lineage viruses as tested by ELISAs against purified viruses. Shown are the quantitative endpoint titers. The negative-control (Neg. Ctrl) antibody used is a MAb directed to influenza A virus H6 HA (MAb 8H9), and the positive control (Pos. Ctrl) is a MAb specific for the influenza B virus NA (MAb 4F11). (B) Neutralization activity of anti-influenza B virus HA MAbs. The MAbs were found to be nonneutralizing in plaque reduction neutralization assays. The IC₅₀ values were determined using Prism (GraphPad) based on the reduction in plaque number. A neutralizing anti-influenza B HA MAb was used as a positive control, and MAb 8H9 (anti-H6) was used as a negative control.

The majority of MAbs target the stalk domain.

The epitopes of neutralizing antibodies can easily be identified through the generation of viral escape mutants in the presence of MAb selection. However, in vitro generation of escape mutants for nonneutralizing antibodies is challenging due to the lack of selection pressure on viral replication. Here, we therefore used different techniques to learn more about the epitopes of the isolated MAbs. One primary question was to distinguish between MAbs that bind to the HA stalk domain and those that bind to the globular head domain. An immunofluorescence (IF) assay was carried out using Madin-Darby canine kidney (MDCK) cells stably expressing a chimeric H8/B HA (cH8/B) on the cell surface, comprising a head domain from an H8 influenza A virus HA (A/mallard/Sweden/24/2002) and a stalk domain from influenza B virus HA (B/Yamagata/16/88). The cH8/B constructs were expressed with alanines at the head-stalk interface (cH8/B_ala) as seen across influenza B virus HAs, or cysteines (cH8/B_cys), as observed in influenza A virus HAs (19). Binding to these constructs would indicate that the stalk is recognized since cross-reactivity between influenza B virus HA and H8 HA globular heads would be highly unlikely due to their low sequence similarity. MAbs KL-BHA-2C6, KL-BHA-2G4, KL-BHA-4C10, KL-BHA-4G12, and KL-BHA-8G3 showed high binding intensity to cH8/B_ala-expressing cells, indicating strong binding to the stalk domain of the HA (Fig. 3). KL-BHA-8G3 binding to the construct with cysteines at the head-stalk interface was of lower intensity than to cH8/B_ala. KL-BHA-4E4, KL-BHA-9B9, and KL-BHA-11C12 were found to bind to a lesser extent to cell lines expressing each cH8/B construct. MAbs KL-BHA-1B5, KL-BHA-1D2, KL-BHA-2H10, KL-BHA-2H11, KL-BHA-3A10, KL-BHA-3H10, KL-BHA-5C5, KL-BHA-6D12, KL-BHA-7C7, KL-BHA-8A5, KL-BHA-8G12, KL-BHA-9C6, and KL-BHA-12F12 showed negligible to no binding toward both constructs. This could be attributed to the fact that they bind to the head domain of the HA, or they bind to conformational epitopes on the stalk of the HA which may not be retained well or accessible in a chimeric construct. Interestingly, a few MAbs mentioned above that do not bind to the cH8/B constructs also have negligible binding to B/Phuket/3073/13 virus, which can be attributed to antigenic drift of the HA. Inversely, MAbs that show low MBCs against B/Phuket/3073/13 also showed moderate to strong binding to the cH8/B constructs.

FIG 3.

Binding of MAbs to cells expressing cH8/B HA. The binding of MAbs to MDCK cells stably expressing cH8/B (H8 head, influenza B virus HA stalk) was tested by immunofluorescence. An anti-H8 MAb, KL-H8-1A7, against the H8 head of the cH8/B construct was used as a positive control, and KL-BNA-4F11, an anti-influenza B NA MAb, was used as a negative control; a secondary-only stain is shown. (A and B) Binding to cells expressing cH8/B_ala (two alanines at the head-stalk interface, as found in influenza B virus HA) (A) and binding to cells expressing cH8/B_cys (two cysteines at the head-stalk interface, as found in influenza A virus HA) (B).

Antibody binding epitopes can be characterized as linear or conformational. Antibodies that bind in Western blots following reducing denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) typically recognize linear epitopes (or microconformational epitopes that refold during incubation of Western blot membranes), while many antibodies that recognize complex conformational epitopes lose their binding activity under these conditions. As such, the binding of each MAb in the panel to recombinant HA protein from B/Lee/40 was assessed via Western blotting. Fourteen of the MAbs bound to linear or microconformational epitopes on the HA, as indicated by the presence of bands on the Western blot (Fig. 4A), with MAbs KL-BHA-1D2, KL-BHA-3H10, KL-BHA-7C7, KL-BHA-8A5, and KL-BHA-9C6 losing binding in the assay.

FIG 4.

The majority of the MAbs bind to linear epitopes on the influenza B virus HA, most of which are located in the stalk domain. (A) Binding to full-length influenza B/Lee/1940 virus recombinant HA under reducing denaturing conditions. MAbs that bound to full-length B/Lee/1940 HA in a Western blot analysis following a reducing SDS-PAGE likely recognize linear or microconformational epitopes on the HA, while an absence of binding indicates that the target epitope is likely conformational in nature and was denatured during the assay. Fifteen of 22 MAbs appear to bind to nonconformational epitopes. An irrelevant H7 HA was used to test for nonspecific binding, and an anti-hexahistidine antibody was used as a positive control. (B) Determination of the binding region for MAbs targeting nonconformational epitopes. MAbs that bound to linear or microconformational epitopes were used to probe against different fragments of the influenza B/Lee/1940 virus HA, as indicated. For reference, the variability across different influenza B virus HAs used in this study is also shown to highlight the highly conserved nature of the stalk domain. The fragments were expressed in HEK293Ts using pCAGGS or pEGFP-C1 expression vectors and tagged with a hexahistidine tag or GFP, respectively, and the cell lysates were used for Western blot analyses. For fragments expressed in pCAGGS (head and stalk III), an anti-hexahistidine tag antibody was used as a positive control, and the control lysate was from HEK 293Ts transfected with pCAGGS empty plasmids. An anti-EGFP antibody was used as a positive control for fragments expressed in the pEGFP-C1 backbone (stalk I, stalk II, and long alpha-helix), and the control lysate was from cells transfected with pEGFP empty plasmids which expressed EGFP protein. (C) Binding to HA pretreated under various pH conditions and/or with a reducing agent. The MAbs displayed different binding profile changes in the ELISA depending on the condition under which the viruses were pretreated: low pH or with DTT, a reducing agent. An increase in binding post-low-pH pretreatment implies that the target epitope is more exposed in the postfusion conformation, while a loss of binding under reducing conditions implies that the target epitope was completely denatured or was located on HA1 which is removed under these conditions. The control MAb CR8059 binds to the head domain, while MAb CR9114 binds to a conformational epitope on the stalk domain. The percent change in area under the curve (from the binding curves generated from absorbance at 490 nm) relative to neutral pH (blue) is charted for each MAb, and the various conditions are indicated (pH 4.4, red; pH 4.4 with 0.2 M DTT, green).

Subsequently, fragments of the influenza B/Lee/1940 virus HA protein were expressed through transient transfections of 293T cells to help determine the region to which MAbs targeting a linear epitope bind. The larger fragments, stalk III (spanning HA2, 21.5 kDa), and HA head (28.1 kDa), were expressed with hexahistidine tags in a pCAGGS expression plasmid, and expression was confirmed with an anti-hexahistidine tag control antibody. Stalk I (peptide sequence following the signal sequence and prior to the globular head domain), stalk II (peptide sequence between the globular head and the stalk domain), and long alpha-helix (a domain in the stalk region highly conserved across influenza B viruses) fragments were expressed in a pEGFP-C1 plasmid N-terminally fused to green fluorescent protein (GFP) to stabilize the smaller fragments. An anti-GFP control antibody confirmed the expression of these fragments (at 32.1 kDa, 33.8 kDa, and 33.6 kDa, respectively), and the control lysate corresponds to 293T cells transfected with empty pEGFP-C1 plasmid. MAbs that bound to linear or microconformational epitopes were used to probe the different HA fragments against the cell lysate in a Western blot (Fig. 4B). None of the 14 MAbs bound to the ∼50-amino-acid peptide sequences of stalk I and stalk II. The majority of the Western blot-active MAbs bound to the HA2 region. The results obtained from the cH8/B binding assay and subunit-based Western blotting are consistent for the majority of MAbs. MAbs with strong stalk binding in the cH8/B IF assay showed a similar behavior in their binding to the long alpha-helix (KL-BHA-2C6, KL-BHA-2G4, KL-BHA-4C10, and KL-BHA-6D12) or outside this region on the HA2 (KL-BHA-4G12). MAbs KL-BHA-1B5, KL-BHA-2H11, KL-BHA-5C5, and KL-BHA-12F12 appear to bind to the globular head domain (and showed no binding in the cH8/B IF described above). MAb KL-BHA-4E4 showed no binding to any of the fragments.

Studying the nature of the antibody-antigen interactions under various pH and reducing conditions is a valuable tool for HA epitope characterization. A change in binding of a MAb to the target epitope under various conditions can attest to the accessibility of the epitope in the pre- or postfusion conformation of the HA, and possibly the location of the epitope on the HA as well. This was achieved through binding assays with HA pretreated under different pH conditions, similar to those carried out by Ekiert et al. (20). Binding of MAbs to B/Yamagata/16/88 purified virus was assessed following pretreatment of the virus-coated plate at neutral pH, pH 4.4, and pH 4.4 in the presence of the reducing agent dithiothreitol (DTT). DTT treatment results in the separation of HA1 and HA2 subunits and allows a determination of whether a MAb binds to the either of the one subunits. Right after treatment, the pH was brought back to neutral for all conditions, and an ELISA was carried out. MAb binding was normalized to the neutral pH condition, and the percent change in area under the curve of binding is represented in Fig. 4C. KL-BHA-2C6, KL-BHA-2G4, KL-BHA-4C10, KL-BHA-4G12, KL-BHA-8G3, and KL-BHA-8G12, all of which bind to HA2 (as seen in Fig. 4B), showed similar binding profile changes. A drop in pH, causing a conformational change of the HA to the postfusion conformation, resulted in increased binding activity of these antibodies, likely because their epitopes are more accessible in the postfusion conformation. The overall improved binding might also hint at the linear nature of the target epitopes which was not disrupted following acidification and reduction. Conversely, for several MAbs (KL-BHA-1B5, KL-BHA-1D2, KL-BHA-2H10, KL-BHA-2H11, KL-BHA-3A10, KL-BHA-3H10, KL-BHA-4E4, KL-BHA-7C7, KL-BHA-7F10, KL-BHA-8A5, KL-BHA-9B9, and KL-BHA-9C6), the majority of which bind to the globular head domain, and to conformational epitopes (as determined by Western blotting), similar or marginally weaker binding was observed at lower pH, which was then completely abolished upon DTT pretreatment. For MAbs that do not bind to the head, this marked reduction in binding following a reduction of disulfide bonds can be explained by loss of conformation or removal of a binding interface. Three of the remaining MAbs showed improved binding during acidification, likely stemming from a better exposure of the epitope, which was then diminished with DTT pretreatment. Minimal binding retention at pH 4.4 with 0.2 M DTT for KL-BHA-5C5 and KL-BHA-11C12 can be explained by residual nondenatured HA1 molecules being present on the plate despite washing due to weak interactions. Overall, the findings from the pH ELISA, taken in conjunction with the Western blotting results and cH8/B IF have allowed us to partially characterize the binding epitopes of these MAbs.

In vivo protection and ADCC induction in vitro.

Recent studies have highlighted the importance of nonneutralizing antibodies in contributing to protection against influenza infection through Fc-mediated effector functions (21, 22). The mechanism by which nonneutralizing antibodies confer protection has been characterized for influenza A virus HAs (23–25), but only limited data are available for MAbs against influenza B virus HA. As mentioned earlier, 22 MAbs were found to be nonneutralizing in vitro against the B/Yamagata/16/88 virus, the HA of which was used as the final immunogen prior to hybridoma fusion. These MAbs were evaluated in a prophylactic setting administered intraperitoneally at 5 mg/kg of body weight in female BALB/c mice 2 h prior to challenge with B/Malaysia/2506/04 (V) virus (Fig. 5A). Interestingly, 12 MAbs conferred complete protection (as defined by 100% survival) in mice receiving a lethal virus challenge; mice in these groups displayed minimal weight loss. Three antibodies were partially protective (as defined by partial survival), and four MAbs displayed no protection. Anti-H6 MAb 8H9 was administered as a negative control; all mice in this group died by day 9 postinfection. MAbs KL-BHA-7F10, KL-BHA-11C12, and KL-BHA-7C7 are not shown because they exhibited weak binding to the HA of B/Malaysia/2506/04, which was used as a challenge virus. Two MAbs each from each category of protective, partially protective, and nonprotective MAbs were selected and used in a similar prophylactic setting against a B/Florida/04/06 (Y) challenge (Fig. 5B). KL-BHA-9C6 and KL-BHA-8G3 were completely protective against lethal challenge. Both partially protective MAbs selected remained as partially protective, while KL-BHA-2G4, a nonprotective MAb in a Victoria-like challenge, was also partially protective when administered prior to a Yamagata-like virus challenge. Overall, similar weight loss and survival profiles were observed across the two influenza B virus (IBV) challenges.

FIG 5.

Prophylactic protection from viral challenge in the mouse model and corresponding in vitro ADCC induction. (A) Five mice per group were given 5 mg/kg of the MAb intraperitoneally 2 h prior to virus challenge with 5 mLD₅₀ of B/Malaysia/2506/04 (V). The MAbs were classified as fully protective, partially protective, or nonprotective based on survival, and the weight loss and Kaplan-Meier survival plots are shown for each group. The dashed line indicates a cutoff of 75% of the initial weight, the humane endpoint. Twelve of 22 MAbs were fully protective in this challenge model. MAb 8H9 (anti-H6 MAb) was used as a negative control at the same dose and is commonly indicated in each group (the control group was shared between the panels). (B) Two MAbs from each category of protective efficacy were selected and administered to mice in a similar prophylactic setting. These mice were challenged with 5 mLD₅₀ of B/Florida/04/06 (Y) virus, and relatively similar weight loss and survival profiles were observed in these mice. (C) In vitro antibody-dependent cell-mediated cytotoxicity reporter assay. The MAbs were tested in an antigen-specific ADCC reporter assay (Promega), and the fold induction of a luciferase-based readout for each MAb was indicated over the negative control (MAb 8H9). The MAbs are divided into the same three groups of fully (filled shapes; left), partially (half-filled shapes; middle), and nonprotective (nonfilled shapes; right) and are indicated with the same symbols as in panel A.

Due to the lack of virus neutralization activity of these MAbs, it is likely that their Fc regions engage corresponding Fc receptors on immune cells in mice leading to protection from mortality and morbidity. Classically, this occurs through antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) (26). Studies have shown that antibodies against the conserved stalk domain are more likely to elicit Fc effector functions in comparison to antibodies interacting with the globular head domain of the HA, specifically against the receptor binding site (23). Favorable contacts of the HA with sialic acid residues on the surface of effector cells, and the Fab region of the antibody with the stalk of the HA along with the Fc-Fc receptor interaction leads to optimal effector functions (24, 25). To test effector function activity in vitro, a commercially available ADCC reporter assay (Promega) was used (Fig. 5C). All antibodies that conferred complete protection in in vivo experiments showed a 20- to 40-fold induction of luciferase readout over the negative control in vitro. The one exception was KL-BHA-4C10, which showed a modest ∼8-fold induction in the ADCC reporter assay. KL-BHA-4C10’s lack of robust in vitro ADCC activity may provide an explanation for the relatively greater postchallenge weight loss observed in the group that received this MAb (Fig. 5A). The fold induction appears to be correspondingly reduced for MAbs classified as partially protective in vivo, while nonprotective antibodies show negligible ADCC induction. The stalk-reactive MAb KL-BHA-2C6, which is partially protective, does not induce ADCC in vitro, and the partial protection conferred could potentially be explained by activating an alternative Fc receptor, as only interactions with mFcγRIV are assessed using this in vitro assay. Finally, MAb KL-BHA-9B9 is an exception that provides no protection but shows activity in the assay at high concentrations. This phenomenon for KL-BHA-9B9 might be explained by low-affinity Fc-Fc receptor (Fc-FcR) interactions.

ADCC induction in vitro correlates with protection from weight loss after viral challenge in vivo.

On average, the in vitro activity of MAbs found to be completely protective in a prophylactic setting at 5 mg/kg in the ADCC reporter assay was higher than for MAbs which were partially or nonprotective (Fig. 6A). To investigate if there was a correlation, the area under the curve (AUC) measured in the ADCC reporter assay was plotted against the average maximal weight loss of each MAb. The average maximal weight loss was calculated by taking the highest percentage of weight loss for a given animal in a group and averaging them across all mice per group. We found a strong statistically significant correlation of ADCC reporter activity in vitro and overall lower weight loss observed in vivo (Fig. 6B). Furthermore, sorting antibodies by isotype revealed that IgG2a isotype MAbs tend to display overall lower weight loss profiles in mice. This is consistent (Fig. 6C and D) when weight loss and ADCC reporter assay activity are stratified by antibody isotype as well. Such results are expected given that IgG2a antibodies have the highest binding affinity for the mFcγRIV receptor, followed by IgG2b antibodies. IgG1 does do not interact well with this receptor (27).

FIG 6.

Correlation between antibody characteristics and in vivo protection. (A) Comparison of ADCC reporter activity in vitro and protective efficacy against challenge in vivo in mice. ADCC activity in vitro (y axis) is graphed against the degree of protection conferred by these MAb prechallenge from the data displayed in Fig. 4. Overall, MAbs that are completely protective in vivo in the prophylactic setting prior to influenza B virus challenge appear to robustly induce ADCC in vitro (**, P = 0.0022). ns, nonsignificant. (B) Correlation of ADCC induction in vitro and weight loss in vivo. The correlation between the in vitro ADCC activity and average (Avg.) maximal weight loss (x axis, in percentage with a 25% cutoff) is shown. There is a strong correlation between a MAb’s ADCC activity in vitro and its ability to protect against weight loss postchallenge. The isotype of the MAbs are indicated: IgG1, blue; IgG2a, red; IgG2b, green. (R² = 0.7567; pattern recognition receptor [PRR] = 0.8699; ****, P < 0.0001). (C and D) Antibody isotype plotted against average maximal weight loss or ADCC induction. On plotting the in vitro ADCC induction and in vivo weight loss for each MAb on the basis of isotype, we find that IgG2a MAbs induce the highest levels of ADCC in vitro, followed by IgG2b and IgG1 MAb(s), and correspondingly also lead to the lowest weight loss observed in our in vivo experiment. (C) **, P = 0.0016. (D) **, P = 0.0019. (E and F) Region of antibody binding compared in context of weight loss or ADCC reporter activity. Instead of antibody isotype, the data sets are represented on the basis of their binding to the head or the stalk of the HA. There is no significant trend in terms of whether the MAbs bind to the head or the stalk of the HA. (G and H) Nature of epitope and its relationship to weight loss or ADCC induction. There is a statistically significant difference between MAbs targeting conformation epitopes and linear/microconformational epitopes in context of inducing higher ADCC in vitro and leading to lower overall weight loss in vivo in the context of viral challenge. MAbs binding conformational epitopes show higher ADCC reporter activity and better in vivo efficacy. (G) *, P = 0.0457. (H) *, P = 0.0476.

Based on the findings from the cH8/B IF and pH ELISA profiles and Western blots against fragments spanning the HA, antibodies were classified as head binding or stalk binding. No statistical significance was observed when the head versus stalk binders were compared for their ADCC reporter activity or in vivo weight loss (Fig. 6E and F). Interestingly, the conformational or linear/microconformational nature of the target epitope appears to influence the in vitro and in vivo functionality of these MAbs (Fig. 6G and H), with MAbs binding to conformational epitopes showing significantly higher ADCC reporter activity and better protection.

DISCUSSION

Classical antibody-based protection from influenza virus-induced morbidity and mortality is associated with neutralizing antibodies that inhibit the interaction between HA and host cell receptors (28). This dogma has been challenged recently by the discovery of MAbs that neutralize virus by inhibiting the fusion between viral and endosomal membranes during entry (HA stalk-reactive antibodies), antibodies that inhibit viral egress (anti-HA stalk, anti-HA head, and anti-NA antibodies), and antibodies that protect via Fc-mediated effector functions (29–32). The vast majority of existing work is focused on influenza A viruses, while little is known about these mechanisms for influenza B viruses (1).

Here, we characterized 22 antibodies that bind broadly to influenza B virus HA. A significant proportion of these antibodies recognized to the HA stalk domain, which is also a target for broadly protective MAbs on influenza A virus HA due to its high conservation (33). While none of the 22 MAbs showed activity in a neutralization assay, a large fraction potently protected mice from lethal challenge with influenza B virus. This protection was most strongly associated with the activity of the respective MAbs in an in vitro ADCC reporter assay that measures engagement with the mFcγRIV receptor. This is reminiscent of the human pan-HA (A and B) MAb CR9114 (12), which does not neutralize influenza B viruses but protects in vivo through Fc-dependent effector functions (25). The ADCC reporter assay can be seen as a proxy for other Fc-mediated effector functions like ADCP as well. In fact, it has recently been shown that macrophages but not neutrophils or natural killer cells are required for protection based on nonneutralizing antibodies in the mouse model (26). Protection and ADCC reporter activity were stronger for IgG2a MAbs than for IgG2b MAbs, as expected based on previously published data, showing that the IgG2a isotype is more actively engaging the activating FcRs (34). The only IgG1 antibody isolated and tested had no protective effect and very little activity in the reporter assay. However, two MAbs, KL-BHA-9B9 and KL-BHA-4C10, were identified as outliers. KL-BHA-9B9 had strong activity in the ADCC reporter assay but no protective effect, while KL-BHA-4C10 was strongly protective but had very weak activity in the reporter assay. It remains to be elucidated on which mechanisms of protection KL-BHA-4C10 depends and why KL-BHA-9B9 is not capable of conferring protection. Additionally, in the context of protection conferred, no strong pattern was seen regarding epitope location (head versus stalk), while MAbs targeting conformational epitopes protected better than MAbs targeting linear/microconformational epitopes.

In summary, we show that on a monoclonal basis in the mouse model, nonneutralizing broadly reactive anti-influenza B virus HA antibodies can afford strong protection against influenza viral challenge. Importantly, this protection is mediated by effector functions but seems to be epitope independent. While the main goal of vaccine strategies is to induce neutralizing antibodies, it might be desirable to optimize future influenza B virus vaccines to induce broadly reactive nonneutralizing antibodies as well.

MATERIALS AND METHODS

Cells, viruses, and recombinant proteins.

Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with Pen-Strep antibiotics mix (penicillin at 100 U/ml, streptomycin at 100 μg/ml; Gibco), 10 ml of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Life Technologies), and 10% fetal bovine serum (FBS; HyClone) was used to passage Madin-Darby canine kidney (MDCK, ATCC #CCL34) cells. Trichoplusia ni medium-formulation Hink (TNM-FH) insect cell medium (Gemini Bioproducts) containing Pen-Strep and 10% FBS was used to grow Sf9 insect cells. High Five cells (BTI-TN-5B1-4 cells; Vienna Institute of BioTechnology clone [35]), used to express recombinant proteins, were propagated in serum-free medium (SFM)-insect cell medium (HyClone). SP2/0 mouse myeloma cells (ATCC CRL-1581) were passaged in complete DMEM as described above, prior to hybridoma fusion. Hybridoma clones produced were initially cultured in ClonaCell-HY medium E (Stemcell Technologies) and progressively switched to culturing in Hybridoma serum-free medium (Hybridoma-SFM; Life Technologies) to facilitate large-scale production.

The A/PR/8/34 (PR8) influenza A backbone virus that expresses the HA of B/Yamagata/16/88 instead of its own HA was described before (18). Influenza B viruses B/Lee/40 (ancestral lineage), B/Great Lakes/1739/54 (ancestral lineage), B/Victoria/2/87 (V), B/Malaysia/2506/04 (V), B/Texas/2/13 (V), B/Yamagata/16/88 (Y), B/Florida/04/06 (Y), and B/Phuket/3073/13 (Y) and the PR8 virus expressing the influenza B virus HA were grown in 8- to 10-day-old embryonated chicken eggs incubated at 33°C for 3 days. For the purposes of mouse adaptation, a naive female BALB/c mouse was infected with 10⁶ PFU of virus and monitored for weight loss. At 3 days postinfection (dpi), the lungs were harvested and homogenized, and the clarified supernatant was used to reinfect another naive BALB/c mouse at a 1:10 dilution. The B/Florida/04/06 and the B/Malaysia/2605/04 viruses were passaged in mice 13 and 5 times, respectively. Both of these viruses were subsequently grown up in eggs, and titers and 50% lethal doses (LD₅₀) were determined. Two mutations in the HA, N212S and I214T, were observed in the mouse-adapted B/Malaysia/2506/04 challenge virus, and a G156E mutation in the HA was observed in the mouse-adapted B/Florida/04/06 challenge virus. Virus preparations for ELISA were made by low-speed centrifugation of allantoic fluid to remove debris (2,000 × g at 4°C for 10 min), followed by ultracentrifugation (at 25,000 rpm for 2 h at 4°C with an SW-28 rotor in a Beckman L7-65 ultracentrifuge) through a 30% sucrose cushion buffered with NTE buffer (100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA balanced to pH 7.4). The supernatant containing allantoic fluid was aspirated, and the pellet was resuspended in phosphate-buffered saline (PBS; Gibco). Baculoviruses for expression of recombinant proteins, including B/Lee/40 HA and B/Yamagata/16/88 HA, were generated as previously described (36). Recombinant HAs were expressed following a detailed published protocol (36, 37).

Hybridoma fusion, antibody isolation, and antibody purification.

One 6- to 8-week-old female BALB/c mouse was inoculated with 10⁴ PFU of B/Lee/40 (ancestral lineage) using the intraperitoneal route. Approximately 4 weeks later, the mouse was intranasally infected with 10⁴ PFU of B/Malaysia/2506/04 (V). Four weeks later, the mouse was injected intraperitoneally with 100 μg of purified PR8 virus expressing B/Yamagata/16/88 HA adjuvanted with 10 μg of poly(I:C). Three days postboost, one mouse was euthanized, and the spleen was harvested. Splenocytes were fused with SP2/0 cells following a published well-described protocol (38). Briefly, polyethylene glycol (Sigma-Aldrich) was used to fuse the harvested splenocytes with SP2/0 mouse myeloma cells, and the clones were propagated on semisolid ClonaCell-HY medium D (Stemcell Technologies). Individual colonies were then transferred to 96-well cell culture plates and grown in medium E for 5 days. Supernatants from hybridoma clones were first screened against recombinant B/Yamagata/16/88 HA and B/Florida/4/06 (Y) HA and then isotyped using a Pierce rapid antibody isotyping kit (Life Technologies), following the manufacturer’s instructions. A secondary screen through ELISAs was carried out against B/Lee/1940, B/Victoria/2/87 (V), B/Florida/4/06 (Y), and B/New Jersey/1/12 (V) purified viruses. Clones that produced IgG isotype antibodies were grown in serum-free Hybridoma-SFM medium to a volume of 300 to 500 ml and incubated for 10 days. The supernatant was harvested by low-speed centrifugation (4,000 × g at 4°C for 10 min) and filtered through a 0.22-μm-pore-size sterile filtration unit (Millipore). The filtrate was applied to a purification column packed with protein G-Sepharose 4 Fast Flow beads (GE Healthcare). Following flowthrough of the supernatant and binding of target MAb to the resin, 3 column volumes of sterile PBS was used for washing and removing nonspecific proteins. Elution buffer (0.1 M glycine [pH 2.7]) was added and incubated on the column for 15 min, and the eluate was collected in a 50-ml Falcon tube containing 5 ml of neutralization buffer (2 M Tris-HCl [pH 9.4]). The MAb was concentrated and buffer exchange to PBS at pH 7.4 was performed. The purified monoclonal antibodies were quantified by measuring the absorbance at 280 nm using the NanoDrop device (Thermo Scientific).

ELISAs.

Ninety-six-well nonsterile flat-bottom Immulon 4 HBX plates (catalog no. 3855; Thermo Scientific) were coated for 16 h (overnight) at 4°C with 2 μg/ml of purified recombinant protein or 5 μg/ml of purified virus at 50 μl/well in 1× coating buffer (SeraCare). The coating buffer was discarded, and plates were blocked with 3% milk powder in PBST (PBS with 0.1% Tween 20; Fisher BioReagents) at 220 μl/well for 1 h at room temperature. For ELISAs to screen hybridoma clones, 50 μl of undiluted supernatant from the cultures was added to the first well, followed by 3-fold (1:3 dilution) serial dilutions in blocking buffer with final volumes of 100 μl per well. The starting concentration for MAbs was 30 μg/ml with 3-fold serial dilutions in blocking buffer. The plates were incubated for 1 h at room temperature, following which they were washed 3 times with 100 μl/well PBST using an AquaMax 4000 microplate washer device (Molecular Devices). The secondary antibody, anti-mouse horseradish peroxidase (HRP)-labeled antibody (Rockland) diluted 1:3,000 in blocking buffer, was added at 50 μl/well, and the plates were again incubated at room temperature for 1 h. The plates were then washed 4 times with intermittent shaking with 100 μl/well of PBST using the plate washer. SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich) was used to develop the plates by adding 100 μl of the substrate solution. The development was allowed to proceed for 10 min, and the reaction was stopped with 50 μl/well of 3 M hydrochloric acid. The plates were then analyzed at an optical density (OD) of 490 nm using a Synergy H1 microplate reader (BioTek). The endpoint titer or minimum binding concentration was defined as the final concentration (dilution) of the antibody at which the signal was greater than a cutoff value (mean with 3 times the standard deviation of all blank wells on a given plate). Cutoff values are calculated independently for each plate. For the binding ELISAs of the MAbs to various viruses, an anti-influenza B NA antibody, 4F11, produced in our laboratory (17), was used as a positive control, and an anti-influenza H6 antibody, 8H9, was used as an irrelevant negative-control antibody.

The pH ELISAs were adapted from a similar technique developed by Ekiert and colleagues (20). The plates were washed twice with 100 μl/well PBST following blocking and incubated with buffered solutions of 300 mM sodium citrate buffer at pH 7.0, pH 4.4, or pH 4.4 with 0.2 M dithiothreitol (DTT) for 30 min before the antibody binding step. The substrate was then brought back to neutral pH prior to incubation with the MAbs. Each antibody was run in duplicate under each given condition. The OD values were analyzed, and the average area under the curve for each MAb under each condition was calculated using a cutoff of the average summed to 3 times the standard deviation of blank wells. The percent change in AUC was calculated for each MAb under each condition in comparison to neutral pH and graphically represented. The final representation of the pH ELISAs were generated using Prism (GraphPad). As control antibodies, CR8059 and CR9114, a broadly cross-reactive influenza B HA head binding MAb, and stalk binding conformational epitope targeting MAb were used (12).

PRNA.

The plaque reduction neutralization assay (PRNA) was performed as follows. Dilutions of MAbs in 1× minimum essential medium (MEM) were incubated with 100 PFU of B/Yamagata/16/88 for 90 min at room temperature with shaking. The incubated solution was then transferred onto a cultured monolayer of MDCK cells in six-well cell culture plates, followed by 1 h of incubation and intermittent rocking every 10 min. The agar overlay applied was supplemented with the corresponding MAb dilutions, and plates were incubated at 33°C for 72 h. Four percent paraformaldehyde (PFA) in PBS was added to each well to fix cells at 4°C for 30 min. The plaques were stained with an anti-influenza B polyclonal serum (from mice infected with B/Lee/1940, B/Malaysia/2506/04, and B/Florida/4/06) at a 1:5,000 dilution in PBS for 1 h at room temperature with shaking. Following washes with PBS, diluted anti-mouse IgG HRP secondary antibody was added to the wells. The plates were developed using TrueBlue reagent (KPL), and the number of plaques was counted. Percent inhibition or plaque reduction was calculated in comparison to a no-antibody control, and the IC₅₀, defined as the concentration of the MAb at which there is 50% plaque number reduction, was calculated on Prism (GraphPad) using a nonlinear regression model. An anti-influenza B virus HA head binding antibody served as a positive control for neutralization, and an anti-H6 antibody, MAb 8H9, was used as a negative control.

Immunofluorescence microscopy.

MDCK cells constitutively expressing cH8/B constructs (19, 39, 40) were cultured in 96-well cell culture plates overnight, following which they were fixed with 3.7% PFA in PBS for 1 h at room temperature. The cells were then washed and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The MAbs were added to their respective wells at a concentration of 10 μg/ml in blocking buffer, with an anti-NA MAb, 4F11 (17), used as a negative control, and anti-H8 MAb 1A7 (41) was used as a positive control. The cells were washed twice with PBS after an hour of primary incubation, and goat anti-mouse Alexa 488 conjugated antibody (Abcam) and 4′,6-diamidino-2-phenylindole (DAPI) both at 1:500 in blocking buffer were added to the cells for secondary staining for 1 h. The plates were washed 3 times with PBS and were then viewed under the EVOS FL cell imaging microscope (Thermo Fisher). The captured images were aligned, processed on ImageJ, and labeled using Adobe Illustrator.

Western blotting.

For the full-length HA Western blotting, recombinant B/Lee/1940 HA and A/Shanghai/1/13 H7 HA at 50 μg/ml were heated to 100°C for 20 min in 2× Laemmli buffer with 2% β-mercaptoethanol (BME). Ten microliters per well of the samples was then loaded onto 4 to 20% gradient polyacrylamide gels (15-well gels; Bio-Rad). SDS-PAGE was performed at 200 V for approximately 30 min until the dye-front exited the gel. The samples were transferred onto nitrocellulose membranes using the iBlot 2 dry blot system (Thermo Fisher) at 20 V for 7 min. For Western blotting, the membranes were blocked for 2 h at room temperature with shaking in 3% milk in PBST blocking buffer. Following this, they were stained with 30 μg/ml of the anti-influenza B virus HA MAb as the primary antibody for 1 h at room temperature with shaking. Subsequently, the blots were washed three times for 5 min each with PBST and stained with a goat anti-mouse IgG–alkaline phosphatase antibody at a 1:10,000 dilution in 1% milk in PBST for 1 h at room temperature. After washing 4 times for 5 min each with PBST, the blots were developed using the alkaline phosphatase (AP) conjugate substrate kit (catalog no. 1706432; Bio-Rad), as per the manufacturer’s protocol, with an initial equilibration step using the 25× color development solution diluted to 1× in double-distilled water (ddH₂O). Recombinantly expressed H7 HA (from H7N9 strain A/Shanghai/1/2013 [42]) was used as a negative control, and an anti-hexahistidine tag antibody was used as a positive control in the Western blotting. The bands were observed at approximately 80 kDa, as expected for a full-length HA protein during SDS-PAGE.

The constructs for the expression of the various subunits spanning the HA, for Western blotting, were designed as follows. Stalk I fragment spanned 42 amino acids (D16 to A57) following the signal sequence, and the head fragment was expressed from N58 to E306. Amino acids A307 to R361 constituted stalk II. G362 to Y552 was classified as stalk fragment III, and D436 to L487 was classified as the long alpha-helix domain. The methionine residue at the start of the signal peptide was classified as amino acid 1. Expression plasmids encoding the various HA subunits were transfected into human embryo kidney 293T (HEK293Ts) cells using the TransIT-LT1 transfection system (Mirus Bio), and the cell lysates were denatured, applied on an SDS-PAGE gels, and transferred onto nitrocellulose membranes which were blocked as described above. Each of the 14 antibodies found to be active in the Western blot analysis against a full-length HA were used as primary MAbs at 10 μg/ml against each of the lysates of cells transfected with the five individual HA fragments. Empty plasmids were used as appropriate transfection and Western blot controls, and antibodies targeting the affinity purification tags (anti-hexahistidine [TaKaRa Bio] for pCAGGS, and anti-enhanced GFP [EGFP; Abcam] for pEGFP-C1 expression vectors) were used as positive controls. An anti-mouse HRP-labeled antibody (Rockland) was used as a secondary antibody at a 1:5,000 dilution in 1% milk in PBST instead of the alkaline phosphatase-tagged antibody. Following this, the blots were washed 4 times for 5 min each with PBST and developed using the Pierce ECL Plus Western blotting substrate (catalog no. 32132; Thermo Fisher), as per the manufacturer’s instructions, and developed on X-ray film (Advansta). Depending on the observed background levels, further washing steps were carried out, and various exposures were captured on film during development. The representative image of each blot was captured. All the blots were aligned and processed as a batch on ImageJ, following which they were labeled using Adobe Illustrator.

ADCC reporter assay.

The in vitro ADCC reporter bioassay kit (Promega) was used to determine the Fc-mediated effector function of the MAbs. Briefly, 30,000 MDCK cells/well were cultured in white-bottom 96-well cell culture plates (Corning) and infected with influenza B/Malaysia/2506/04 virus at a multiplicity of infection (MOI) of 5 overnight in the absence of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin. The following day, the medium was aspirated, and 25 μl/well of antibody dilutions were added except in blank wells. Twenty-five microliters per well of assay buffer, Roswell Park Memorial Institute medium (RPMI 1640; Gibco) medium supplemented with low-IgG serum (Promega), was added to each well, along with 25 μl/well of ADCC effector cells (75,000 cells/well). Blank wells received 25 μl/well of assay buffer instead of the MAb dilutions. The plates were incubated for 6 h at 37°C with 5% CO₂, followed by the addition of 75 μl luciferase assay substrate (Promega) to each well, and the luminescence readout was captured using a Synergy H1 microplate reader (BioTek). The fold induction over the negative-control antibody (anti-H6 HA MAb, 8H9) was plotted for each MAb, with the concentration represented on a logarithmic scale. AUC values were calculated and plotted in Prism 7.0 (GraphPad).

In vivo mouse challenge study.

Female 6- to 8-week-old BALB/c mice (5 per group) were injected intraperitoneally with 5 mg/kg of purified MAb 2 h prior to infection. B/Malaysia/2506/04 or B/Florida/04/06 was used as a challenge virus at 5 murine 50% lethal doses (mLD₅₀) administered intranasally, and weight loss was observed over 14 days. Control mice received anti-H6 antibody, MAb 8H9, at the same dose and time point. After 14 days, all mice were euthanized, and the weight loss and Kaplan-Meier survival plots were generated based on the obtained data. For the correlational analysis, the maximum weight loss for each mouse in a group was averaged. If all the mice were euthanized prior to day 14, 25% was the designated average maximal weight loss for that group. Complete protection was defined as 100% survival, while partial protection involved a fraction of the mice surviving in a group. The correlation analyses of the average maximal weight loss and survival, with isotype, nature of epitope, binding region, and more importantly, in vitro ADCC induction, were generated using Prism (GraphPad). All the challenged mice were euthanized, having lost 25% of their initial weight, the established humane endpoint as per established IACUC protocols, or at day 14 postchallenge. All in vivo experiments were conducted in accordance with the guidelines of the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee.

Statistical and phylogenetic analysis.

A one-way analysis of variance (ANOVA) was used to determine statistical significance in comparing in vitro ADCC induction of MAbs classified as nonprotective, partially protective, and fully protective, and for comparison of different antibody isotypes in context of ADCC activity or weight loss. Pearson correlation analysis was used to determine the Pearson correlation coefficient and coefficient of determination for the in vitro ADCC activity and in vivo weight loss. Standard unpaired t-tests were applied to analyze other pairwise comparisons. All statistical analyses were performed in Prism (GraphPad). For phylogenetic analysis, sequences were procured from the Global Initiative on Sharing All Influenza Data (GISAID) or the Influenza Research Database (FluDB), aligned preliminarily in the MEGA7 software for quality control. The final alignment was performed using Clustal Omega, and the output was then used to generate a phylogenetic tree using the FigTree software. The generated tree was annotated and highlighted using Adobe Illustrator. For the sequence alignment, sequences were aligned using the MEGA7 software, and the regions of variability were mapped onto the HA sequence at the appropriate sites. The final annotation and image processing were carried out on Adobe Illustrator.