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Published in final edited form as: Virology. 2016 Nov 5;500:178–183. doi: 10.1016/j.virol.2016.10.024

Influenza A Virus Hemagglutinin Specific Antibodies Interfere with Virion Neuraminidase Activity Via Two Distinct Mechanisms

Ivan Kosik 1, Jonathan W Yewdell 1
PMCID: PMC5127735  NIHMSID: NIHMS828341  PMID: 27825034

Abstract

We studied the ability of monoclonal Abs (mAbs) recognizing the major hemagglutinin (HA) antigenic sites to inhibit neuraminidase (NA) cleavage of sialic acids on fetuin. We show that virion associated-NA activity in the enzyme linked lectin assay (ELLA) is largely dependent on HA-mediated attachment of virions to immobilized fetuin. For a Sb-specific mAb, there is a nearly perfect correlation between neuraminidase inhibition and blocking virus attachment to immobilized fetuin. By contrast, Sa-, Ca-, and Cb- specific mAbs block NA activity in ELLA or the traditional thiobarbituric acid assay by sterically interfering with NA access to substrate. We conclude first, that ELLA with intact virus can only be used to measure anti-NA Abs if serum lacks HA-specific Abs, and second, that anti-HA Abs block NA activity by both limiting virion interaction with sialic acid containing surfaces and by sterically limiting NA access to sialic acids attached to macromolecules.

Keywords: influenza, ELLA, TBAA, HA antibodies, canonical antigenic sites, NA inhibition

Introduction

Influenza A virus (IAV) remains a serious human pathogen, exerting enormous health and economic costs. Virions contain two oligomeric surface glycoproteins, hemagglutinin (HA, trimeric) and neuraminidase (NA, tetrameric). Each recognizes terminal sialic acid (SA), but with opposite activities. HA attaches virus to SA on the target cell surface to initiate the infectious cycle, while NA releases virions from cell surface SA to complete the infectious cycle (Gottschalk et al., 1959; Palese et al., 1974) The functions of HA and NA must mesh to balance attachment vs. release (Banks et al., 2001; Das et al., 2013; Heaton et al., 2013; Hensley et al., 2011; Mitnaul et al., 2000; Yano et al., 2008).

The rapid antigenic evolution of HA and NA (“antigenic drift”), necessitates frequent vaccination of the human population with updated strains. The effectiveness of influenza vaccination is evaluated by levels of antibodies (Abs) specific for HA, and less often, NA. Anti-HA Abs are of paramount importance since they directly neutralize viral infectivity by blocking cellular attachment and or fusion, and confer potent protection in vivo (Reading and Dimmock, 2007). NA Abs reduce IAV replication by preventing virion release from the infected cell surface, limiting infection to a single infectious cycle (Kilbourne et al., 1968; Seto and Rott, 1966). NA-specific Abs can, however, protect in vivo, and since NA evolves more slowly than HA, is receiving increasing attention as a vaccine target (Eichelberger et al., 2016, Sylte and Suarez, 2009). While NA is present in most vaccines, vaccine potency is strictly based on HA content and immunogenicity.

NA is generally 5- to 10-fold less abundant on virions than HA (Hutchinson et al., 2014), and is believed to reside in clusters of 3 to 5 oligomers scattered among a forest of HA spikes (Harris et al., 2006). Given the close proximity of HA and NA on the virion surface, it might be expected that Abs specific for one of the glycoproteins affect the function of the other. Indeed, decades ago, serum HA Abs were shown to inhibit NA activity, most probably by steric hindrance (Paniker, 1968; Russ et al., 1974). Such Abs might provide enhanced neutralization capacity by inhibiting viral release from infected cells. More generally, the effect of HA-specific Abs on NA function should provide insight into the organization of HA and NA on virions (Harris et al., 2006).

Two major assays are used to measure Ab-mediated NA inhibition (NI). The thiobarbituric acid assay (TBAA) chemically measures SA released from fetuin, an inexpensive and highly sialylated 48 kDa glycoprotein. Due to greater safety, simplicity, and throughput, the enzyme linked lectin assay (ELLA) has largely replaced the TBAA (Couzens et al., 2014; Lambre et al., 1990; Sandbulte et al., 2009). In the ELLA, NA release of SA is measured by lectin binding to the now exposed penultimate galactose in an N-linked oligosaccharide, typically attached to fetuin, a relatively inexpensive glycoprotein abundant in fetal calf serum (Couzens et al., 2014).

Here we use a panel of well characterized mAbs that bind one of the four canonical antigenic sites (Sa, Sb, Ca, Cb, Figure 1) (Caton et al., 1982) on the HA of A/Puerto Rico/8/34 (H1N1) to explore how anti-HA Abs interfere with NA activity as measured by TBAA or ELLA. Our data provide insight into both how HA facilitates NA activity, and demonstrate that anti-HA Abs interfere with NA activity via two distinct mechanisms.

Figure 1. Canonical antigenic sites of the HA molecule.

Figure 1

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We used Pymol software to visualize canonical epitopes on a surface representation of the HA molecule as determined by x-ray crystallography. (A) Side view of the HA trimer with Cb site (red) and Ca site (teal). (B) Top view of the HA globular head showing Sa (orange) and Sb (blue) antigenic sites. Sialic acid is shown in bright green

Results and Discussion

Using ELLA, we first compared the NI activities of the anti-HA mAb panel (Figure 2, Table 1). As with a representative anti-NA mAb used as a positive control (NA2-1C1), each of the mAbs exhibited NI (Fig. 2A), with a characteristically sharp drop occurring over a 10-fold range, consistent with simple binary law of mass action binding. Ab specificity is shown by the lack of NI activity exerted by a negative control mAb specific for M1 protein (M2-1C6). Although the HA-specific mAbs display a wide range of IC50s, their efficiency at blocking NI (NI efficiency in Table 1), calculated as the mAb equilibrium dissociation constant KD, (determined by ELISA) divided by the Ab concentration required for 50% NI (IC50), is narrowed to a 10-fold range when we normalize for mAb binding affinities, which vary up to 200-fold (Table 1), to account for antibody occupancy on HA.

Figure 2. Comparison of TBAA vs. ELLA.

Figure 2

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We measured NI activity for HA Abs recognizing different canonical HA antigenic sites by ELLA and TBAA in the absence and presence of TX100. (A) ELLA in absence of TX100, (B) TBAA in absence of the TX100, (C) ELLA in presence of TX100, (D) TBAA in presence of TX100. We used the NA specific mAb NA2-1C1 as a positive control for NI. We used a no virus control (NVC) to determine the assay background values. We used the signal measured for the negative control M1-specific M2-1C6 mAb to normalize the data, which are expressed as % NA activity. For pdmH1N1, we measured NI for H17-L10 in the absence and presence of the TX100 in ELLA (E) and TBAA (F).We used GraphPad Prism6 software to fit the data a nonlinear regression curve. Error bars indicate the standard deviation (SD). We tested each sample in duplicate, and show the average of two independent experiments.

Table 1.

Functional Activities of mAbs.

Ab KD [nM] IC50ELLA [nM] IC50TBAA [nM] AI50 [nM] NIELLA Effic NITBAA Effic AI Effic
Y8-1A6 (Sa) 0.56 0.56 0.48 < 1.10 26% < 42%
IC5-4F8 (Sb) 0.04 0.04 0.09 < 0.09 10% < 36%
H17-L10 (Ca) 8.12 8.12 1.78 229.4 6.66 =100% =100% =100%
H9-D3 (Cb) 0.73 0.73 0.40 150 2.13 40% 14% 25%
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We determined the KD and IC50 values for the mAbs shown empirically and calculated the efficiency (Effic) of NA inhibition (NI) and attachment inhibition (AI) by dividing the KD values by the respective IC50s to determine activity per bound Ab. We then relate activity values as the % of H17-L10 activity, which demonstrates the highest efficiency in all of the assays. Y8-1A6 and IC5-4F8 did not attain 50% inhibition in the TBA assays, so their IC50 values are shown as <.

Adding 0.5% TritonX-100 (TX100) to release HA and NA from virions increased the efficiency of NA2-1C1 (anti-NA) mediated-inhibition by 14-fold. By distinct contrast, TX100 completely abrogated the NI activity of all HA-specific mAbs (Fig. 2C). This indicates that NI activity mediated by anti-HA Abs requires the physical proximity of HA and NA, and is not trivially due to mAb cross-reactive binding to NA or non-specific factors present in the mAb preparations.

To broaden these findings to a more recent human isolate, we examined the NI activity of the Ca-specific mAb H17-L10 on A/California/07/2009 (H1N1) (pdmH1N1) (H17-L10 is the only mAb in the panel that binds pdmH1N1). H17-L10 exhibits significant NI in ELLA vs. pdmH1N1 (Fig. 2E), and once again, NI activity is completely abrogated by adding TX100 to dissociate virions.

We next examined the NI activity of HA-specific mAbs in the TBAA. MAbs specific for Ca and Cb sites maintain their NI activity (Fig. 2B) relative to NA2-1C1 (though all of the mAbs demonstrated an approximate 100-fold decreased efficiency in TBAA vs. ELLA). As with the ELLA, NI activity was eradicated by detergent treatment (Fig. 2D). H17-L10 recapitulated these properties when tested with pdmH1N1 (Fig. 2F).

In contrast to Ca- and Cb-specific mAbs, Sa- and Sb-specific mAbs exhibited more limited NI activity in TBAA, inhibiting 25% or less NA activity at the highest concentrations used (Fig. 2B). The Sb-specific mAb demonstrated a particularly large decrease in NI activity in TBAA vs. ELLA.

How to explain the greater capacity of Sa and Sb mAbs to inhibit NA in ELLA vs. TBAA? After incubating virus and mAbs with fetuin coated plates, and washing away free virus, we directly measured IAV binding to plate bound-fetuin in the ELLA using the small fluorogenic NA substrate 4-(methylumbelliferyl)-N-acetylneuraminic acid (muNANA) to detect virions via their NA activity. Importantly, due to the small size muNANA, none of the HA mAbs we tested inhibits NA cleavage of muNANA. This is expected, since even NA2-1C1 (and many other anti-NA Abs) fail to inhibit cleavage of such small substrates (Fazekas de St Groth, 1963). After recording the values, we then washed away muNANA and completed the normal ELLA.

Superimposing muNANA and ELLA Ab titration curves (Fig. 3) revealed a near perfect correlation for the IC5-4F8 (Sb) inhibition of virus binding and neuraminidase activity, consistent with the conclusion that its NI activity is nearly entirely based on blocking viral attachment, and not sterically blocking the access of fetuin to the NA active site. This is consistent with the low activity of IC5-4F8 in the TBAA (Table 1).

Figure 3. Role of Viral Attachment in ELLA.

Figure 3

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We modified the standard ELLA assay by measuring virus attachment using a fluorescent NA substrate as a virus-autoreporter, removing unbound virus by washing. After removing muNANA, we continued the ELLA on the same wells. We tested each sample in duplicate and show the average of two independent experiments: error bars indicate the SD. We used GraphPad Prism6 to calculate the area under the curve (AUC) from fluorescent kinetics data for each Ab concentration, and plotted it vs. ELLA NA activity as % of attachment and NA activity in absence of Ab (A, B, C, D). NVC, no virus control. NA activity in the absence (blue) or presence (red) of TX100 detergent as determined using ELLA (E) or muNANA (as in panel E) (F). To determine possible allosteric effect of virus attachment to NA activity, we incubated virus dilutions in fetuin vs. BSA coated plates. We used muNANA to measure NA activity of fetuin bound vs unbound virus (G). We used GraphPad Prism6 software to generate a correlation plot for measured NA activities.

By contrast, each of the other mAbs tested demonstrated significant inhibition of ELLA activity when virus remained attached to plate bound-fetuin. This can be quantitated in the differences in IC50s for inhibiting virion binding to fetuin vs. NA activity. The increased efficiency of blocking ELLA NA activity vs. virus attachment ranges from 2.3-fold for Y8-1A6 (Sa), 3.7-fold for H17-L10 (Ca), and 5.3-fold for H9-D3 (Cb).

Based on the ability of anti-HA Abs to inhibit NA ELLA activity by blocking virus attachment, we predicted that HA-mediated virus attachment to plate bound-fetuin enhances virion NA activity. Indeed, treating virus with TX100 to dissociate HA from NA reduces NA ELLA activity 100-fold (Fig. 3E), having no effect on NA activity measured by muNANA (Fig. 3F).

Could the enhanced NA activity of fetuin bound-virus result from conformational changes in NA that increase intrinsic enzyme activity measured by cleavage of muNANA? To test this, we added graded amounts of virus to 96-well plates coated with either fetuin or a non-glycosylated negative control protein (bovine serum albumin (BSA)), and allowed virus to bind at 4 °C before measuring NA activity with muNANA at 37 °C (Fig. 3G). This revealed no significant difference in NA activity between bound (fetuin) and unbound (BSA) virus (control samples showed that > 70% of input virus bond to fetuin coated-wells, while essentially no virus binds to BSA coated-wells, as determined measuring muNANA activity of bound virus or virus present in the supernatant).

We conclude that virus binding to fetuin does not alter intrinsic NA enzymatic activity, but rather that the enhanced NA activity in ELLA exhibited by intact virus is due to enhanced access to immobilized fetuin via HA-mediated attachment. This NA-enhancement phenomenon seems likely to extend to virus bound via HA to cells and other SA-coated surfaces in vivo.

Our findings extend classical studies showing that HA-specific Abs inhibit neuraminidase activity in the TBAA (Paniker, 1968; Russ et al., 1974). We show that this is a particular technical issue for ELLA, an extremely robust assay that has replaced the classical TBAA. Our findings emphasize the necessity to use an indicator virus in ELLA that has a HA mismatched with the sera being tested (Couzens et al., 2014). This can be accomplished by choosing an HA from different subtype, or simply by using TX100 or other mild detergents to dissociate virus.

We show that anti-HA antibodies inhibit NA activity by two distinct mechanisms:

  • Blocking binding of virus to surface bound NA-substrate.

  • Sterically inhibiting NA access to large substrates, either in solution or when immobilized.

Anti- HA mAbs display a range of NI activities in ELLA based on each mechanism. The Sb- specific mAb tested acts exclusively by blocking virus attachment, while the Cb specific mAb acts mostly by steric inhibition of NA access to SA on fetuin. The Sa- and Ca-specific mAbs we tested utilize both mechanisms. The extent to which these differences in mAb activity are due to their relative locations of their cognate epitopes on the HA spike will be the subject of a future study.

Material and Methods

Viruses

We propagated IAV strains A/Puerto Rico/8/1934 (H1N1) (PR8) and A/California/07/2009(H1N1) (pdmH1N1) in the allantoic cavity of 11 d embryonated chicken eggs, collected allantoic fluid 48 h p.i, and stored aliquots at −80°C.

Abs

All Abs used are mouse IgG mAbs and have been described previously (Brooke et al., 2013; Gerhard et al., 1981; Magadan et al., 2013).

ELISA Ab Avidity Measurement

We determined mAb avidity for HA via ELISA. We coated ELISA plates (96-well, Greiner Bio-One) with 50ng purified IAV PR8 or pdmH1N1 diluted in DPBS (100μl per well). After overnight incubation at 4°C, we washed plates three times with DPBS supplemented with 0.05% Tween-20. We diluted Abs to 100nM in 1% BSA in DPBS. We diluted Abs 5-fold, and added to plates for 90 min at room temperature. After extensive washing with DPBS+0.05% Tween-20, we detected bound Abs by incubating plates with HRP-conjugated rat anti- mouse IgG kappa chain (SouthernBiotech) for 1 h at room temperature in the dark. We washed ELISA plates again with DPBS+0.05% Tween-20 and incubated with SureBlue TMB Microwell Peroxidase Substrate (KPL) for 5 min at room temperature. We stoped enzymatic reaction by adding HCl to inactivate HRP and convert the chromophore to a more detectable species. We determined the A450 on a Synergy H1 plate reader (Biotec), and calculated the Kd from dilution curves using GraphPad Prism 6 software to fit one site binding.

NI Assays

We performed the ELLA as described (Couzens et al., 2014) with minor modifications. In place of coating buffer, we used DPBS, we avoided 0.5% Tween-20 in all steps to preserve virion integrity. We determine the optimal amount of PR8 or pdmH1N1 virus by performing ELLA with serially diluted virus over an 80-fold range and then used virus amounts yielding an A450 between 2 and 2.5. We coated ninety-six-well ELISA plates (Greiner Bio-One) with 100 μl DPBS fetuin solution (25μg/ml) overnight at 4°C. We diluted Abs to 100 nM and then we diluted Abs serially 5 fold. We pre incubated 50 μl of Ab dilutions with 50 μl virus in sample diluent (DPBS, 1%BSA) for 30 min at room temperature and then we added samples to fetuin coated plate for 18-22 h at 37°C. We washed plates extensively with washing buffer (DPBS, 0.05% Tween-20) and then we added 100 μl peanut-HRP (Sigma Aldrich) diluted 1000 fold in sample diluent for two hours at room temperature in the dark. After washing, we added 10μl of SureBlue™ TMB Microwell Peroxidase Substrate (KPL) to each well for 5-10 min in the dark and combined with 100μl 1M HCl to stop the enzymatic reaction. Then we used a Synergy H1 plate reader to measure the A450 (Biotec). To liberate HA and NA from virion we added 0.5% TritonX-100 in sample diluent for 10 min at room temperature, and then followed the standard ELLA. We set the signal of M2-1C6 (anti M1 mAb) as 100% NA activity. We plotted data with GraphPad Prism6 software and fit nonlinear regression curves using the dose response inhibition model.

We performed the miniaturized TBAA as described (Sandbulte et al., 2009). After titrating virus to determine the optimal working dilution, we incubated 5 μl virus with 5 μl 3.17 fold dilutions of purified mAbs (starting at 1μM) in Micro Amp optical 96-Well plates (Life technologies). After 30 min at room temperature, we added 5 μl fetuin (25 mg/ml) and incubated sealed plates 18-22 h at 37°C. We then added 5 μl of sodium periodate, incubated for 15 min at room temperature, and added 25 μl arsenite solution. After extensive vortexing, we added 50 μl thiobarbituric acid and incubated for 15 min at 99°C then cooled samples to 4°C, using a thermal cycler. We added 100 μl of acid butanol to extract the chromophore and measured A550 with a Synergy H1 plate reader (Biotec). We set the signal obtained with M2-1C6 (anti M1 mAb, negative control) as 100% NA activity. To liberate HA and NA from virion we added 0.5% Triton X100 to virus for 10 min at room temperature, and then followed the standard TBAA protocol. We plotted the data with GraphPad Prism6 software and fit nonlinear regression curves using the dose response inhibition model.

Attachment inhibition linked ELLA

To simultaneously measure the effect of mAbs on both attachment and neuraminidase activity in the ELLA, we coated black, half area high binding ninety six-well plates (Nunc) with 50μl fetuin in DPBS (25μg/ml) overnight at 4°C. We serially diluted 6.67 nM Abs 3.1 fold and mixed with 200-fold diluted allantoic fluid and incubated for 30 minute. We then added 50 μl of Ab-virus mixture to washed fetuin coated plates, incubated for 7 h at 37°C, and removed free virus by washing 6-times with DPBS. We then added 50μl of 200μM 4- (methylumbelliferyl)-N-acetylneuraminic acid (muNANA) in 33mM MES pH 6.5 with4mM CaCl2 and recorded fluorescence (Ex=360nm, Em=450nm) at 37°C for 1 hour at 1 minute intervals using a Synergy H1 plate reader (Biotec). We then washed plates extensively with DPBS, 0.05% Tween-20 and incubated with 50 μl of peanut-HRP (Sigma Aldrich) diluted 1000-fold in sample diluent for two h at room temperature in the dark. After washing, we added 50 μl of SureBlue™ TMB Microwell Peroxidase Substrate (KPL) to each well, incubated for 5-10 min in the dark, and then added 50μl 1M HCl to terminate enzyme activity. We measured the A450 with a Synergy H1 plate reader at 450 nm (Biotec), and used GraphPad Prism 6 software to generate the area under the fluorescence kinetic curves. The signal in the absence of mAb defines 100% attachment/NA activity. We expressed AUCs and ODs as % attachment or NA activity and plotted against given Ab concentration. We fitted nonlinear regression curves using the dose response inhibition model with GraphPad Prism/

MuNANA NA activity

To measure influence of NA HA dissociation, we serially diluted in presence or absence of 0.5% TX100 for 10 min at RT and then combined 25μl of virus with 25μl of 200μM 4-(methylumbelliferyl)-N- acetylneuraminic acid (muNANA) in 33mM MES pH 6.5, 4mM CaCl2, recording fluorescence (Ex=360nm, Em=450nm) at 37°C for 1 hour at 1 minute intervals by a Synergy H1 plate reader (Biotec). To measure the influence of virus attachment to fetuin on NA activity, we coated black, half area high binding ninety-six-well plates (Nunc) with 50μl fetuin (25μg/ml) or 1% BSA in DPBS overnight at 4°C. After washing, we serially diluted freshly purified virus 1.25 fold (initially 4.25pg/well) and added to wells in 25ul of 33mM MES pH 6.5, 4mM CaCl2. After 4 h at 4°C, we added 25μl muNANA in 33mM MES pH 6.5, 4mM CaCl2 to wells and recorded fluorescence as described above. We generated fluorescence kinetics curves for each virus dilution and calculated the AUD using GraphPad Prism. To determine the influence of attachment on NA activity, we calculated AUCs for given dilutions of fetuin bound virus, and plotted against respective AUCs of virus dilutions incubated in BSA coated wells. Pearson's analysis was used to determine correlation.

Highlights.

Anti-HA antibodies inhibit NA activity by two distinct mechanisms:

  • Blocking binding of virus to surface bound NA-substrate.

  • Sterically inhibiting NA access to large substrates, either in solution or when immobilized.

To avoid interference from anti-HA antibodies, ELLA must use either detergent treated virus to dissociate glycoproteins or virus with serum-mismatched HA.

Acknowledgements

We thank Glenys Reynoso for providing outstanding technical assistance. This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases.

Footnotes

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