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Contents lists available at ScienceDirect

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Measuring the neutralization potency of influenza A virus hemagglutinin stalk/stem-binding antibodies in polyclonal preparations by microneutralization assay

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Wenqian He a,1, Caitlin E. Mullarkey a,1, Matthew S. Miller b,⇑

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a

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Department of Biochemistry and Biomedical Sciences, Institute for Infectious Diseases Research, McMaster Immunology Research Centre, McMaster University, Hamilton, Ontario, Canada b

a r t i c l e

i n f o

Article history: Received 11 March 2015 Received in revised form 29 April 2015 Accepted 30 April 2015 Available online xxxx Keywords: Influenza A virus Broadly neutralizing antibodies Hemagglutinin Universal vaccine Microneutralization Polyclonal

a b s t r a c t The discovery of broadly-neutralizing antibodies that bind to the hemagglutinin stalk/stem domain has opened exciting new avenues for the development of ‘‘universal’’ influenza virus vaccines and therapeutics. Unlike strain-specific antibodies which bind to the hemagglutinin head domain and inhibit receptor binding, antibodies that bind to the stalk domain function to inhibit later stages of infection. The hemagglutination inhibition assay has long been the standard for evaluating titers of neutralizing hemagglutinin-specific antibodies in serum. The assay has the beneficial properties of being relatively rapid, easy-to-perform, and requires very little specialized equipment. Historically, hemagglutination inhibition titers of 40 or above against a given strain of influenza has been considered a correlate of protection on a population level. Unfortunately, this assay cannot be used to measure titers of hemagglutinin stalk-specific antibodies due to their lack of hemagglutination inhibiting activity. This has necessitated the development of novel reagents and assays capable of sensitive and specific detection of broadly-neutralizing HA stalk-binding antibodies in polyclonal mixtures. Here, we describe a novel microneutralization-based assay that utilizes recombinant influenza A viruses expressing chimeric hemagglutinin molecules and ‘exotic’ neuraminidase to measure titers of broadly-neutralizing antibodies in polyclonal preparations. Ó 2015 Published by Elsevier Inc.

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1. Introduction

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The recent discovery of broadly neutralizing antibodies (bnAbs) recognizing the conserved stem/stalk region of hemagglutinin (HA) represents a game-changing discovery in the field of influenza virus vaccines and therapeutics. Vaccination has long served as the primary approach to combating both seasonal and pandemic influenza virus outbreaks. However, current vaccines are limited in the protection they confer. Seasonal vaccines elicit a narrow strain specific response and generate antibodies predominantly to the HA globular head domain. Consequently, the ‘holy grail’ of vaccinologists has long been the successful induction of heterosubtypic immunity, or protection against all influenza A viruses (IAVs)

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⇑ Corresponding author at: Department of Biochemistry and Biomedical Sciences, Institute for Infectious Diseases Research, McMaster Immunology Research Centre, MDCL 2324, McMaster University, 1280 Main St. W, Hamilton, ON L8S 4K1, Canada. E-mail address: [email protected] (M.S. Miller). 1 Equal contribution.

regardless of subtype or strain, a concept first described by Schulman and Kilbourne in 1965 [1]. This new class of bnAbs directed against the conserved HA stalk has offered hope that a vaccine eliciting such cross-protective responses may soon be a reality. This would constitute a significant breakthrough in public health as influenza viruses remain among the most formidable pathogens in the modern world. From a therapeutic standpoint these findings are also promising as such antibodies have the potential to treat severe infections and to be used prophylactically in high-risk groups. Based on HA phylogeny IAVs can be classified into two groups. Ekiert and colleagues described the first human monoclonal antibody capable of binding a highly conserved region in the HA stem, which was able to bind most group 1 HAs [2]. Shortly thereafter the same group also reported on a second antibody that neutralized most group 2 HAs [3]. While the neutralization activity of HA stalk-binding antibodies is typically limited to either group 1 or group 2 viruses, in rare instances antibodies capable of binding both group 1 and groups 2 HAs have been isolated from human

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plasma cells [4]. To date several groups have succeed in isolating broadly neutralizing antibodies from both mice and humans [2,3,5–9]. In the past five years several studies have demonstrated that in humans bnAbs can be boosted by natural infection or vaccination [10–18]. Vaccination regimens have also proven capable of boosting bnAbs in animal models [19–21]. However, these are usually generated to a lower extent as compared to antibodies against the globular head. Moreover, monoclonal bnAbs appear to neutralize less potently than antibodies which bind the HA head [22–25]. Interestingly, recent evidence has emerged to suggest that the observed difference in the potency of antibodies recognizing the HA head as compared to the conserved stalk is greatly reduced in a polyclonal setting [25]. Moreover, HA stalk-specific IgA antibodies have proven to be particularly potent in terms of their quality and magnitude [25]. Although further work is needed to dissect out the mechanisms by which HA stalk-binding IgAs achieve greater neutralization potency, these data are particular encouraging in light of universal influenza vaccine initiatives. While characterization of bnAbs has brought the exciting prospect of a universal influenza vaccine into reach, it has also poised a new set of challenges in the laboratory. Chief among these is sensitive detection of bnAbs and their neutralization capabilities. HA stalk-binding antibodies achieve neutralization through fundamentally different mechanisms than strain-specific antibodies which bind to the HA head domain. While these mechanisms have not been fully elucidated, stalk antibodies have been shown to inhibit viral egress, interfere with the fusion of viral and host membranes, inhibit HA maturation and engage in Fc–Fc receptor interactions [23,25–27]. Conversely, classical antibodies recognizing the HA head domain block binding of the influenza virus HA to sialylated cell surface receptors. Such antibodies can be measured readily by hemagglutination inhibition assays. It is widely accepted in the field that the standard surrogate marker for protection is an HI titer P 40 [28]. However, defining a correlate of protection for HA stalk-binding antibodies is decidedly more problematic and has yet to be defined. Fortunately a number of novel reagents have been produced that have helped to surmount these obstacles. Chimeric HA (cHA) molecules that consist of stalk and head domains derived from different influenza A subtypes have been generated [29]. The stalk domain for these cHAs consists of an H1 or H3 stalk domain (strains currently circulating in humans) in combination with an exotic globular head domain to which the vast majority of humans are naïve. In addition to recombinant expression in insect cells, functional influenza viruses expressing cHAs can also be rescued. These viruses exhibit growth properties similar to wild-type viruses and can be propagated in embryonated chicken eggs [29]. Importantly these proteins and viruses allow for specific measurement of stalk-binding antibodies in polyclonal mixtures as they remove the interference of antibodies which bind to the globular head. As a result, they have become critical tools in both immunological and virological assays. Specifically, the use of chimeric HA molecules as substrates in a standard ELISA assays has permitted the quantification of bnAb titers in serum samples. This method has been successfully applied in evaluating universal vaccination strategies in animal models [20,30,31], in human vaccine studies [12–14] and in longitudinal analyses of antibody compartment [11]. However, it is important to keep in mind that while ELISAs can be used to gauge binding of HA stalk-binding antibodies, this technique does not provide any functional information about the neutralizing potential. As HA-stalk binding antibodies lack hemagglutination inhibition (HAI) activity and are known to neutralize downstream of receptor binding, it is necessary to utilize an assay allowing for multicycle replication. Microneutralization

(MNT) assays are best suited for these purposes. In terms of measuring HA head-binding antibodies, it has been shown that MNT assays offer greater specificity and sensitivity than HAI assays [32]. Antibodies directed against the other influenza virus surface glycoprotein, neuraminidase (NA), have also been shown to be protective in animal models [33]. Consequently the generation of influenza viruses expressing cHAs coupled with an ‘‘exotic’’ NA protein has allowed for the development of MNT protocols to measure the ability of HA stalk-binding antibodies to neutralize in vitro [12–14,20,25,30,31].

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2. Materials and methods

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2.1. Serum inactivation

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Prior to use in MNT assays, serum must first be inactivated to remove non-specific inhibitors. Human serum can be inactivated by incubation at 56 °C for 30 min. Serum derived from other animals should be inactivated by receptor destroying enzyme (RDE) or trypsin-heat-periodate treatment.

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2.1.1. Cholera filtrate/RDE treatment Reconstitute Cholera filtrate (Sigma, C8872) in 5 ml sterile water as directed. Dilute to 10 in calcium saline, pH 7.2. Add four volumes of 10 RDE to 1 volume of serum and incubate overnight (18–22 h) at 37 °C. Add five volumes of 1.5% sodium citrate solution, pH 7.2 per volume of serum. Heat at 56 °C for 30 min to inactivate remaining RDE.

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2.1.2. Trypsin-heat-periodate treatment Add 0.5 volumes of 8 mg/ml TPCK-treated trypsin (Sigma, T1426) to 1 volume of serum and heat at 56 °C for 30 min. Cool to room temperature (RT), and then add 3 volumes of 0.011 M metapotassium periodate solution per volume of serum, incubating for 15 min at RT. Add 3 volumes of 1% glycerol saline solution and incubate at RT for another 15 min. Finally, add 2.5 volumes of 0.85% saline, at which point serum is ready for use.

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2.2. Polyclonal immunoglobulin G (IgG) purification

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In addition to the direct assessment of HA stalk-binding bnAbs in serum, the assay is also amenable to measurement of bnAbs from purified polyclonal antibody preparations. Polyclonal IgG can be purified from serum over Protein G or Protein A-based affinity columns (for example, Protein G Sepharose 4 Fast Flow, GE Healthcare, 17-0618-01). While conditions must be optimized based on column and reagent type, a general procedure is described here. Dilute serum 1:4 in PBS and filter to remove any large particulate that may clog the column. Apply to column and allow draining by gravity flow (check manufacturer’s instructions to determine column capacity). Wash column thoroughly with 3 column volumes of phosphate buffered saline (PBS) to remove any unbound protein. Elute bound IgG using one column volume of 0.1 M Glycine/HCl buffer, pH 2.2. Neutralize immediately by eluting directly into 0.1 column volumes of 2 M Tris–HCl, pH 10. Purified antibodies can then be concentrated and dialyzed against PBS by a variety of methods, including centrifugal filters (ex. Amicon Ultra-15 centrifugal filter units, Millipore, UFC903008).

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2.3. Selection of recombinant cHA-expressing viruses

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The selection of appropriate cHA-expressing viruses is crucial to the sensitivity and specificity of this assay. In general, two main classes of viruses are used: those expressing group 1 HA stalk domains and those expressing group 2 HA stalk domains. Details

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regarding the construction of these viruses has been reported previously [29]. The most important consideration is to ensure that serum/antibody samples do not exhibit HAI activity against the cHA-expressing virus being used to measure HA stalk-binding bnAb titers. This can usually be achieved by using cHA constructs with head domains from ‘‘exotic’’ influenza virus subtypes to which humans are not usually exposed. However, HAI assays should be performed to definitively demonstrate a lack of activity. Since antibodies against the NA protein can also contribute to virus neutralization, the use of 6:2 reassortant viruses expressing ‘‘exotic’’ NA proteins (in the A/Puerto Rico/8/34 (PR8) background) can further enhance the specificity of the assay [12]. We have found that the N3 NA from A/Swine/Missouri/4296424/06 to be particularly well-suited for this purpose [12]. Viruses carrying N3 NA have not circulated in humans, and therefore we do not find neuraminidase inhibiting antibodies in human polyclonal sera that would interfere with the specific detection of HA stalk-binding antibodies. To compare HA stalk-specific antibody responses to those directed against a particular HA protein, 6:2 reassortants expressing a given wild type HA can be used (Fig. 1).

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2.4. Preparation of MDCK cells

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Madin-Darby Canine Kidney (MDCK, ATCC CCL-34) cells should be plated in 96-well plates such that they are 70–90% confluent at the time of inoculation and in log-phase growth. MDCK cells should be cultivated in DMEM supplemented with 10% fetal bovine serum (FBS), 1 Penicillin/Streptomycin (Pen/Strep) and 25 mM HEPES.

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2.5. Determination of virus input

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Prior to performing the MNT assay, the virus must be titered such that the signal-to-noise ratio is maximized. This can be done

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by determining the 50% tissue culture infectious dose (TCID50) of the virus using the Reed–Muench method (as described in [34], or by simply testing the number of plaque-forming units (pfu) per well that results in optimal signal-to-noise ratio. We have found that 100 TCID50/50 ll, or 102 to 103 pfu/well is ideal for most viruses [12,25]. It is essential that infection conditions are identical to those that will be used during the MNT assay. See Section 2.7 for instructions on performing cell-based ELISA.

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2.6. Microneutralization assay

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In an empty 96-well plate, add the starting concentration of serum or antibodies to wells A1-A10. Wells A11 and A12 should be left blank for use as positive and negative controls. Serum/antibodies should be diluted serially down each column such that the final volume per well is 50 ll. Dilutions should be performed in DMEM supplemented with 1 Pen/Strep, 25 mM HEPES and 1 lg/ml TPCK-treated trypsin (Sigma, T1426). The appropriate dilution series to use in MNT assays may differ between serum samples and purified antibody preparations. When assessing antibody titers in serum, two fold serial dilutions typically generates a suitable dilution curve. Starting at a 1:10 dilution also allows for comparison to results from HAI assays. However, when assessing purified antibody preparations, it may be necessary to employ a dilution series ranging from two to ten-fold. Dilute virus to the empirically-determined working concentration (as determined in Section 2.5) and add 50 ll to each well in columns 1–11. Column 11 will serve as the ‘virus-only’ control. Column 12 serves as the ‘uninfected’ control and should receive 50 ll of TPCK-trypsin supplemented growth medium only (Fig. 2). Incubate mixtures of virus + serum/antibodies for 1 h at 37 °C.

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Cal/09 N3

cH5/1 N3

Cal/09 HA

vRNA

N3

cH5/1 HA

Fig. 1. Use of cHA-expressing viruses to quantify HA stalk-binding bnAbs in polyclonal preparations. To compare strain-specific anti-HA antibody titers to HA stalk-binding antibody titers, 6:2 reassortants can be used. One virus should express the wild type HA molecule of interest (ie. the Cal/09 N3 virus), while HA stalk-specific antibodies should be assessed using a cHA-expressing virus with an appropriate group 1 or group 2 HA stalk domain and ‘‘exotic’’ HA head domain (ie. cH5/1 N3). To eliminate the contribution of NA-directed antibodies, the viruses should also express exotic NA molecules from a non-human IAV.

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any non-infectious virus particles. Re-incubate cells at 37 °C, 5% CO2 overnight (or approximately 18 h). In some cases, a longer 44–48 h incubation period may also work. Since HA stalk-binding bnAbs function post-attachment, it is necessary for them to remain present throughout the course of multi-cycle virus replication in order to fully gauge their neutralization potency. After overnight incubation, remove spent media and wash cells with PBS. To fix, add pre-chilled (to 20 °C) 80% acetone diluted in PBS. Incubate at 20 °C for 20 min. Remove fixative and allow plate to dry.

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1hr @ 37°c

2.7. Cell-based ELISA

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24-48hr @ 37°c

The readout for this assay is a cell-based ELISA to detect and quantify expression of influenza A virus nucleoprotein (NP). Begin by blocking cells with 5% non-fat milk diluted in PBS for 30 min at RT. Remove blocking buffer and quench with 3% hydrogen peroxide (in PBS), incubating for a further 20 min at RT. Wash three times with PBS/0.01% Tween-20 (PBS-T). Dilute primary antibody (anti-NP, biotin conjugated; EMB Millipore MAB8257B) 1:2000 in 5% non-fat milk. Add 50 ll of primary antibody dilution to each well and incubate for 1 h at RT. At the end of this incubation, repeat plate washing three times with PBS-T before adding HRP-conjugated streptavidin (EMB Millipore 18-152) diluted 1:5000 in 5% non-fat milk. Plate should be allowed to incubate for 1 h at RT before washing three times with PBS-T. After the final wash, add 100 ll of HRP substrate (SigmaFAST OPD) to each well and incubate for 5–10 min at RT (signal in virus control wells should be strong, but not saturated). At this point, stop the reaction using 3 M HCl and read optical density (OD) at 490 nm using an appropriate plate reader. 2.8. Data analysis

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Data from the MNT assay can be expressed in a variety of ways, however the most common would be to report a 50% MNT titer (MNT50), which is best for serum samples. To do this, first, calculate the average OD of the uninfected wells, and then subtract this value from the OD readings of each sample-containing well. This will correct for non-specific background signal. Next, to calculate MNT50 titers, use the following equation to determine the MNT50 cutoff:

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Two-fold

2. Add virus to the plate

3. Transfer the serum or to MDCK cell plate

4. Fix cells and ELISA S1

S2

S3

S4

S5

VC CC

Fig. 2. Schematic representation of MNT assay.

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Following the incubation, remove growth media from MDCK cells and wash three times with PBS. Add virus/antibody mixture to MDCK cells and incubate for 1 h at 37 °C, 5% CO2 to allow for adsorption. During incubation, prepare a second 96-well plate containing identical dilutions of antibody as were originally mixed with virus, but this time add only growth media supplemented with TPCK-treated trypsin. After adsorption, remove virus/antibody mixture from MDCK cells, wash three times with PBS and transfer media containing diluted antibodies. This step removes



ðaverage of OD of virus only wellsÞ  ðaverage OD of uninfected wellsÞ 2

10 20 40 80 160 320 640 1280 Average MNT50 threshold

Sample 1

Sample 2

RAW

Norm

0.276 0.353 0.563 0.715 0.72 0.846 0.768 0.782

0.000 0.007 0.217 0.369 0.374 0.500 0.422 0.436

a

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or

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ðaverage of Column 11Þ  ðaverage of Column 12Þ x¼ 2

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Table 1 MNT50 determination from human serum samples against cH5/1 N3 virus. Reciprocal serum dilution

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Sample 3

Sample 4

RAW

Norm

RAW

Norm

RAW

Norm

0.263 0.321 0.386 0.640 0.800 0.873 0.891 0.867

0.000 0.000 0.040 0.294 0.454 0.527 0.545 0.521

0.301 0.359 0.480 0.785 0.817 0.931 1.044 0.863

0.000 0.013 0.134 0.439 0.471 0.585 0.698 0.517

0.325 0.341 0.478 0.656 0.856 0.856 0.902 0.839

0.000 0.000 0.132 0.310 0.510 0.510 0.556 0.493

BOLD indicates well at which MNT50 is reached. a Norm indicates background-subtracted OD values.

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Infection control

Cell control

0.81 0.886 0.918 0.87 0.973 1.013 0.959 0.844 0.909 0.282

0.357 0.344 0.347 0.353 0.346 0.346 0.35 0.328 0.346

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% Neutralization

Purified Polyclonal IgG Sample 1 Sample 2 Sample 3 Sample 4

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[54.71] [45.39] [74.51] [38.09]

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2

1

0

log [antibody] (µg) Fig. 3. Determination of HA stalk-binding bnAb IC50 from purified human IgG. Polyclonal IgG from 4 human donors was purified over a protein G columns. An MNT assay was then performed using cH5/1 N3 virus to determine the IC50 of HA stalk-binding bnAbs in each preparation. After adsorption, plates were incubated for 44 h prior to fixation and ELISA. Data represents the mean of technical duplicates. [#] indicate IC50 values as calculated using Graphpad Prism in lg/ml.

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All background-normalized values below ‘x’ should be considered positive for neutralization. As for HAI assays, reciprocal dilutions may also be reported (Table 1) [12,34]. Alternatively, 50% inhibitory concentration (IC50) values can be calculated after plotting neutralization curves with purified antibody preparations using statistical software [25].

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3. Results

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MNT assays were performed against cH5/1 N3 virus using serum (Table 1) and purified IgG (Fig. 3) from four human donors who exhibited HAI titers P 40 against A/California/04/09 (exposure to which is known to boost HA stalk antibody titers). Each donor was found to have an MNT50 titer of 40 when serum samples were analyzed (Table 1). IC50 values were determined for purified IgG preparations from the same donors. This readout is substantially more sensitive than the MNT50 determination, but results were consistent with IC50 value for all donors clustering between 38.09 and 74.51 lg/ml. Therefore, the approximately 2-fold difference in IC50 between the lowest and highest donor would not necessarily be expected to result in differences in MNT50 titer, which is sensitive only to differences greater than twofold.

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4. Discussion

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The discovery of bnAbs that bind to the HA stalk domain has ushered in a new era for influenza virus vaccine and therapeutics development. While the characterization of monoclonal antibodies initially set the field into motion, it has quickly been realized that the elicitation of polyclonal bnAb responses is of critical importance. Given the unique mechanisms through which HA stalk-binding antibodies function, novel tools and techniques are required to assess the magnitude and potency of such polyclonal responses. The description of chimeric HA molecules and viruses expressing these constructs has made it possible to sensitively measure bnAbs recognizing the HA stalk and their neutralization capabilities. MNT-based assays, in particular, have proven to be especially powerful tools in assessing the functional neutralizing potency of HA stalk-binding antibodies. As immunization strategies to induce stalk Abs progress into the clinic, techniques utilizing cHAs and chimeric viruses will not only allow for the evaluation of candidate vaccines, but will additionally help to define a correlate of protection. It is important to keep in mind, however, that in vitro assays may fail to capture certain effector functions at play in vivo. More specifically, it has been shown that

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HA stalk-binding antibodies mediate protection in vivo through Fc-dependent functions, such as antibody-dependent cellular cytotoxicity [23]. Nevertheless, in working toward novel vaccines and therapies that rely on HA stalk bnAbs, rapid in vitro assays that are amenable to high-throughput analysis will be essential in evaluating the magnitude and efficacy of responses.

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Acknowledgements

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The authors would like to thank Peter Palese for the generous donation of reagents. This work was supported by funding from McMaster University to M.S.M.

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stem-binding antibodies in polyclonal preparations by microneutralization assay.

The discovery of broadly-neutralizing antibodies that bind to the hemagglutinin stalk/stem domain has opened exciting new avenues for the development ...
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