viruses
ArticleStructure-Guided Creation of an Anti-HA Stalk Antibody F11
Derivative That Neutralizes Both F11-Sensitive and -ResistantInfluenza A(H1N1)pdm09 Viruses
Osamu Kotani 1, *,† , Yasushi Suzuki 2,† , Shinji Saito 3, *,† , Akira Ainai 3 , Akira Ueno 3 , Takuya Hemmi 3 ,Kaori Sano 3 , Koshiro Tabata 3,4 , Masaru Yokoyama 1 , Tadaki Suzuki 3 , Hideki Hasegawa 2 and Hironori Sato 1
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Citation: Kotani, O.; Suzuki, Y.; Saito,
S.; Ainai, A.; Ueno, A.; Hemmi, T.;
Sano, K.; Tabata, K.; Yokoyama, M.;
Suzuki, T.; et al. Structure-Guided
Creation of an Anti-HA Stalk
Antibody F11 Derivative That
Neutralizes Both F11-Sensitive and-Resistant Influenza A(H1N1)pdm09
Viruses. Viruses 2021, 13, 1733.
https://doi.org/10.3390/v13091733
Academic Editor:
Stacey Schultz-Cherry
Received: 30 July 2021

Center for Pathogen Genomics, National Institute of Infectious Diseases, Tokyo 208-0011, Japan;
yokoyama@nih.go.jp (M.Y.); hirosato@nih.go.jp (H.S.)
Center for Influenza and Respiratory Virus Research, National Institute of Infectious Diseases,
Tokyo 208-0011, Japan; yasuzuki@nih.go.jp (Y.S.); hasegawa@nih.go.jp (H.H.)
Department of Pathology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;
ainai@nih.go.jp (A.A.); a-ueno@nih.go.jp (A.U.); t-hemmi@nih.go.jp (T.H.); ka–sano@nih.go.jp (K.S.);k-tabata@czc.hokudai.ac.jp (K.T.); tksuzuki@nih.go.jp (T.S.)
Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University,
Hokkaido 001-0020, Japan
Correspondence: konioo@nih.go.jp (O.K.); ssaito@nih.go.jp (S.S.)
Osamu Kotani, Yasushi Suzuki and Shinji Saito contributed equally to this work.

Abstract: The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes
the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational
rearrangements of HA for virus infection. Here we characterized molecular interactions between
the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand thestructural basis of the actions and modifications of this antibody. In silico structural analyses usinga model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical
properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic
cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked
suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structureguided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for theF11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivativethat can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise
the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered
conformational rearrangements and suggest that in silico guiding experiments can be useful to createanti-HA stalk antibodies with new phenotypes.

Accepted: 26 August 2021
Published: 31 August 2021

Keywords: influenza virus; anti-HA stalk antibody; molecular interactions; molecular dynamics
simulation; antibody modification; neutralization assay

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1. Introduction
Infection with the influenza virus often causes serious respiratory diseases in humans,
and thus presents a persistent public health threat. Although vaccination is a promisingstrategy to suppress the magnitude of an influenza epidemic, current vaccines rarely
overcome the genetic diversity of influenza viruses. The major immunodominant region
for raising neutralization antibodies is located in a highly variable head domain of envelope
glycoprotein hemagglutinin (HA) [1–4]. Consequently, neutralization antibodies raised
against the head domain are usually strain-specific, and often drive the selection of newvariants that are less sensitive to the circulating antibodies [5]. Therefore, antibodies
that target more conserved regions have been sought to combat infection by influenza
viruses [6–8].

Viruses 2021, 13, 1733. https://doi.org/10.3390/v13091733

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Monoclonal antibodies that target the stalk domain of the HA protein have beenshown to protect mice against a broad range of influenza viruses [9]. Moreover, viruses
that acquired resistance to the anti-HA stalk antibodies were attenuated in mice and could
be controlled with a candidate vaccine [10]. The stalk domain is located in an extracellular
portion of the HA protein, and is a key structural unit for the pH-dependent conformationalchanges of the HA protein to gain membrane fusion activity for genome uncoating in theendosome, thereby playing essential roles in establishing virus infection in the cells [11].
The stalk domain is relatively more conserved within and among influenza virus strainsin nature, as compared with the HA globular region [2,4]. This may imply that the stalk
domain is under strong functional/structural constraints against changes. Thus, antibodies
targeting the stalk domain can be beneficial tools to combat infections of highly mutableinfluenza viruses [6–8]. To date, many anti-HA stalk antibodies have been isolated andcharacterized for their binding modes to HA protein [9,12–17]. Interestingly, about halfof these antibodies bind to HA in a manner that crosslinks two HA monomers of the
HA trimer on the virion [9,12–14]. Based on the structural information, the anti-HA stalk
antibodies have been proposed to inhibit conformational changes of the HA protein that
establish the infections of target cells [9,12–17].
Recently, we independently isolated an anti-HA stalk antibody, termed F11, froman individual inoculated with an intranasal inactivated influenza vaccine [18]. In its IgA
form, F11 was able to neutralize the H1N1, H3N2, and H5N1 strains of influenza A, but
in its IgG form it had lower cross-reactivity [18]. We also isolated variants of influenza
A(H1N1)pdm09 that exhibited reduced susceptibilities to neutralization mediated by
the F11 antibody [18]. We found that single substitutions near the hydrophobic cleft of
the HA stalk region could confer F11 resistance to the virus in association with changes
in the physicochemical properties of the hydrophobic cleft [18]. However, the binding
mode of F11 to HA and therefore the molecular mechanisms underlying the F11-mediatedneutralization remain unknown. To address each of these issues, we herein examined thestructural basis of molecular interactions between the F11 Fab fragment and glycosylated
HA trimer using in silico techniques involving molecular dynamics (MD) simulations.
We then used the obtained information to create a F11 variant that can neutralize both
F11-sensitive and F11-resistant influenza A(H1N1)pdm09 viruses.
2. Materials and Methods
2.1. Molecular Modeling of the Trimeric HA Ectodomain of Influenza A(H1N1)pdm09
A three-dimensional model of the HA trimer ectodomain of the A/Narita/1/2009(H1N1)pdm09 virus in the ligand-free state was first constructed by the homology modelingmethod using the reported amino acid sequence of HA of A/Narita/1/2009 (H1N1)pdm09 [19](Figure S1). The Molecular Operating Environment (MOE) (Chemical Computing Group
Inc., Montreal, QC, Canada) was used for the homology modeling. A high resolution
crystal structure of an HA trimer of the A/California/04/2009 (H1N1) virus (PDB code:
3LZG, a resolution of 2.6 Å) [20] was used as the modeling template. The template
represents a structure of the extracellular portion of the H1N1 HA strain. The highmannose oligosaccharide Man5 GlcNAc2 was attached to potential N-glycosylation sites in
HA using tools in GLYCAM-Web [21]. Subsequently, the glycosylated HA trimer model
was subjected to MD simulation as described for HA trimers of influenza A virus [18,22].
Briefly, the simulations were performed using the pmemd.cuda.MPI module in the Amber
16 program package [23] with the ff14SB force field for simulation of protein [24], with the
GLYCAM06j-1 force field for simulation of glycan [25], and with the TIP3P water model
for simulation of aqueous solutions [26]. A non-bonded cut-off of 10 Å was used. Bond
lengths involving hydrogen were constrained with SHAKE, a constraint algorithm thatsatisfies Newtonian motion [27]. The time step for all MD simulations was set to 2 fs. After
heating calculations performed for 20 ps up to 310 K using the NVT ensemble, simulations
were executed for 400 ns in 150 mM NaCl using the NPT ensemble (at 1 atm, 310 K).

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2.2. Docking Simulations of the Glycosylated HA Ectodomain and Anti-HA Stalk Antibody F11
A three-dimensional model of the stalk antibody F11 Fab fragment was constructedusing the Antibody Modeler application of MOE [28]. Amino acid sequences of theantibody F11 immunoglobulin light chain (GenBank accession number: QII15722) andheavy chain (GenBank accession number: QII15721) were used for modeling of the Fabfragment (Figure S1A) [18]. The obtained models were optimized by energy minimization
using the Amber10: Extended Huckel Theory (EHT) force field implemented in MOE.
Docking simulations were performed using the Dock application of MOE [29,30]. Briefly,
amino acid residues in the HA stalk domain (HA1: 18–57; HA2: 345–515) (Figure S1B)
from a structure of the glycosylated HA trimer at 400 ns of MD simulation were defined
as a receptor for binding of stalk-antibody F11, while an energy minimized structure of avariable region of the F11 Fab fragment was defined as a ligand for binding to the HA stalk
domain of the glycosylated-HA trimer. Subsequently, physicochemically possible docking
poses between the receptor and ligand moieties were comprehensively searched with theDock application. The docking simulations were executed to collect 100 possible docking
poses. Binding energies of the F11 Fab fragment to the glycosylated-HA trimer ectodomain
were calculated with the Dock tool using individual docking poses.
2.3. MD Simulation of the Glycosylated HA Trimer Docked to the F11 Fab Fragment
The complex model obtained by in silico docking simulation was subjected to MD simulation under the conditions described previously with HA trimers of influenza A virus [18,22],
which were essentially the same as the conditions used for MD simulations of HIV-1 structuralproteins such as capsid [31] and envelope gp120 bound to CD4 and CCR5 [32]. Briefly, MD
simulations were performed using the pmemd.cuda.MPI module in the Amber 16 program
package [23] with the ff14SB force field for simulation of protein [24], with the GLYCAM06j-1
force field for simulation of glycan [25] and with the TIP3P water model for simulation of
aqueous solutions [26]. A non-bonded cut-off of 10 Å was used. Bond lengths involvinghydrogen were constrained with SHAKE, a constraint algorithm that satisfies Newtonian
motion [27]. The time step for all MD simulations was set to 2 fs. After heating calculations
performed for 20 ps up to 310 K using the NVT ensemble, simulations were executed for
200 ns in 150 mM NaCl using the NPT ensemble (at 1 atm, 310 K).
2.4. Calculation of Root Mean Square Deviation (RMSD)
Calculations of RMSDs were done as described previously [32,33]. Briefly, RMSDs
between the heavy atoms of the initial complex structure and the structures at given time
points of the MD simulation were calculated to monitor the overall structural changes
during the MD simulations using the cpptraj module in AmberTools 16 [23].
2.5. Calculation of Root Mean Square Fluctuation (RMSF)
The trajectory files during last 20 ns of MD simulations (n = 10,000) were used to
calculate RMSF. RMSF of the Cα atoms of amino acid residues was calculated to obtain
information regarding the atomic fluctuations of individual amino acid residues of HA
protein during MD simulations as described previously [18,31–33] using the tools in Amber
16 [23]. Briefly, the average structures during the last 20 ns of MD simulations were used
as reference structures for RMSF calculation. RMSF, which quantifies differences between
average values and those obtained at a given time point of MD simulation, was calculatedusing the ptraj module, a trajectory analysis tool in AmberTools 16 [23].
2.6. Analysis of Noncovalent Interaction Sites and Networks between HA and F11
Analyses of noncovalent intermolecular interactions for F11 antibody binding to the
HA protein during MD simulations were performed as described previously [33]. Briefly,
noncovalent interactions between the F11 antibody and HA protein were identified using the Contact Analysis application of MOE. This application allows identification of
distance-dependent noncovalent interactions between atoms, such as hydrogen bonds,

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Van der Waals interactions, π-interactions, and ionic interactions. HA-F11 complex structures (n = 20) collected at every 1 ns during 180 to 200 ns after MD simulations wereused for the search of interaction sites. Noncovalent interaction networks in the HA/F11
complex detected in the 20 complex structures were visualized using Cytoscape software
version 3.8.2 [34], a program for integrated models of biomolecular interaction networks.
2.7. Protein Patch Analysis
Assessment and visualization of hydrophobic patches on the HA-F11 complex model
were done as described previously [31] using the Protein Patch Analyzer tool in MOE [35–38].
Briefly, protein hydrophobic patches with a minimum area of 50 Å2 for protein-protein
interactions [39,40] were identified with the HA-F11 complex structure at 200 ns of MD
simulation using the Protein Patch Analyzer.
2.8. Shannon Entropy Analysis
Amino acid variation at contact sites of the HA protein was analyzed with Shannon
entropy as described previously [31,41]. Amino acid sequences of the HA protein of theinfluenza A virus H1 (n = 36,564; former seasonal H1N1 and H1N1pdm) and H3 (n = 53,989;
H3N2 and H3N2v) were obtained from the GISAID database [42]. Shannon entropy wascalculated on the basis of Shannon’s equation [43]:H (i ) = − ∑ p( xi ) log2 p( xi )( xi = G, A, I, V, . . .)xi

where H(i), p(xi ), and i indicate the amino acid entropy score for an individual position,
the probability of occurrence of a given amino acid at that position, and the number ofthe position, respectively. An H(i) score of zero indicates absolute conservation, whereas ascore of 4.4 bits indicates complete randomness.
2.9. In Silico Site-Directed Mutagenesis of the F11 Antibody
F11-HA complex structures obtained during 180 to 200 ns after MD simulations wereused for site-directed mutagenesis of the F11 antibody. For the comprehensive mutagenesis
study, F11 residues forming noncovalent bonds with HA glycan or amino acid wereidentified with the structures during 180 to 200 ns of MD simulation using the Contact
Analysis application of MOE. Subsequently the contact residues and surrounding residuesof F11 were individually substituted with 19 nonself amino acid residues. Modeling of
the F11 mutants and calculation of changes in the F11 stability and binding affinity to HA
by the mutations were done using the Protein Design application of MOE, along with the
MM/GBVI program to calculate the binding affinity as described previously [44–46]. The
results were expressed by the change in the free energy of binding (∆∆G). In some cases,
binding energies of individual complexes of F11-HA obtained during MD simulations
were calculated using the Potential Energy application of MOE to assess the distribution ofbinding energies in solution.
2.10. Cells and Viruses
Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection; CCL34) were maintained at 37 ◦ C/5% CO2 in minimum essential medium (MEM; Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific,
Waltham, MA, USA) and pen-strep mix (100 units/mL penicillin and 100 µg/mL streptomycin; Life Technologies). Humanized MDCK (hCK) cells [47] were maintained at 37 ◦ C/5%
CO2 in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM; Sigma-Aldrich, St. Louis,
MO, USA) containing 5% fetal bovine serum, pen-strep mix (100 units/mL penicillin and100 µg/mL streptomycin), 5 µg/mL blasticidin (InvivoGen, San Diego, CA, USA), and2 µg/mL puromycin dihydrochloride (Life Technologies). Expi293F cells (Thermo Fisher
Scientific) were maintained at 37 ◦ C/8% CO2 in Erlenmeyer cell culture flasks (Corning,
Tewksbury, MA, USA) containing Expi293 Expression Medium (Thermo Fisher Scientific).

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A/Narita/1/2009 (H1N1)pdm09 virus [19] and A/Narita/1/2009 (H1N1)pdm09-derived
F11 escape mutants (C1 and G6 [18]) were propagated in MDCK cells.
2.11. Site-Directed Mutagenesis, Expression, and Purification of IgG Antibodies
Plasmids encoding F11 γ1HC and F11 κLC were prepared as previously described [18].
DNA fragments encoding the variable region of the heavy or light chain of each mutant
F11 clone were Homo sapiens codon-optimized and synthesized by the GenPlus Highthroughput Gene Synthesis service (GenScript, Piscataway, NJ, USA). Synthesized DNA
fragments were cloned into γ1HC or κLC vectors and used for the site-directed mutagenesis. To generate IgG1, Expi293F cells grown in Expi293 Expression Medium were diluted
to 2.5 × 106 cells/mL and transfected with 26 µg of γ1HC and 24 µg of κLC per 50 mL offinal culture volume using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). At 7 days post-transfection, cell culture supernatants were centrifuged at 1200× g
and filtered to remove cell debris. The supernatants were then purified using CaptureSelectCH1-XL (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purified
antibodies were concentrated using Amicon Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off of 30 kDa; the buffer was changed to 20 mM phosphatebuffer (pH 7.4) using a Zeba Spin Desalting Column (Thermo Fisher Scientific).
2.12. Assessment of Mutated IgG1 Antibody Quality by SDS-PAGE Analysis
To confirm successful production and purification of mutated IgG1 antibodies, purified
antibodies were analyzed by SDS-PAGE on 5–20% gradient e-PAGEL gel (ATTO Inc.,Tokyo, Japan). Precision Plus Protein Dual Color Standards (Bio-Rad, Hercules, CA, USA)
were used as molecular weight markers. The SDS-PAGE gels were stained with QuickCBB PLUS (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the
manufacturer’s instructions.
2.13. Microneutralization (NT) Assay
The microneutralization assay was performed using hCK cells [47] and 100 TCID50 of
influenza virus, essentially as previously described [48,49]. Briefly, each sample (seriallydiluted 2-fold) was mixed with an equal volume of diluent containing influenza virus(equivalent to 100 TCID50 ) and incubated for 30 min at 37 ◦ C. This mixture was added
to a monolayer of hCK cells in the wells of a 96-well plate. Four control wells containing
virus or diluent alone were included on each plate. The plates were incubated for 5 days
at 37 ◦ C/5% CO2 . All wells were observed for the presence or absence of cytopathic
effects under light microscopy, fixed with 10% formalin phosphate buffer for more than
5 min at room temperature, and then stained with Naphthol blue black. After washingand drying, cells were solubilized with 0.1 M NaOH and absorbance (A) was read at
630 nm. The average A630 value was determined from virus-only controls (A virus) andmedium only controls (A cell). Values > 50% of the specific signal, calculated using theformula X = (A cell − A virus)/2, were considered positive for neutralization. NT activity
was defined as the reciprocal of the lowest concentration (µg/mL) of antibody, at which
point A630 was >X.
2.14. Statistical Analysis
All statistical analyses were performed using the Prism statistical software package(version 7.0; GraphPad Software). An ordinary one-way ANOVA followed by Dunnett’s
multiple comparison test or a Kruskal–Wallis test followed by Dunn’s multiple comparison
test was used to analyze each dataset, as indicated in the figure legends. The threshold forstatistical significance was set at 5% (p < 0.05).

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3. Results
3.1. Molecular Modeling of the Glycosylated HA Trimer Ectodomain Docked to the F11 Fab Fragment
To conduct an in silico structural study of the interactions between the F11 antibody
and HA, we constructed a molecular model of the glycosylated HA trimer ectodomain
docked to the F11 Fab fragment using homology modeling, molecular dynamics (MD)simulation, and docking simulation as described in Materials and Methods. We first
constructed a model of the unliganded HA trimer ectodomain of the A/Narita/1/2009(H1N1)pdm09 virus [19] used in a previous report [18] and the present study by homology
modeling. The high-mannose oligosaccharide Man5 GlcNAc2 was added to the potential
N-glycosylation sites in the trimeric HA using tools in GLYCAM-Web [21].
To obtain trimeric HA ectodomain structures in solution, the initial model was subjected to MD simulation. The structural changes after the start of MD simulation weremonitored with root mean square deviation (RMSD) [23] between the initial model structure and the structures at given time points of the MD simulation as described for HA
trimers of influenza A virus [18,22] (Figure 1A). The RMSDs sharply increased in the beginning and reached a near plateau after 20 ns of the MD simulations. The results suggestthat the structural distortions in the initial HA trimer model were relieved shortly after the
onset of MD simulation, and reached a state of thermodynamic equilibrium in solution.
The MD simulation also disclosed that glycans at the HA stalk domain had partially hidden
the hydrophobic groove [50] located near the binding interface of the reported anti-HA
stalk antibodies [9,12–14,51,52] (Figure 1B). These results suggest that the HA ectodomain
in solution preserves a structure that is partially protected against anti-HA stalk antibodies
by the HA stalk glycans.
Using the HA trimer model in the equilibrium state under solution conditions, we
conducted in silico docking simulation between HA and the F11 Fab fragment (Figure 1C).
The distribution of the free energy of binding (∆G) showed that various docking poses
were physicochemically possible. Among the top 100 docking poses, the vast majorityof F11 Fab was bound to a region near the hydrophobic groove [50], in the same manner that anti-HA stalk antibodies were previously shown to bind to the trimeric HA
ectodomain [9,12–14,51,52] (Figure 1D). We then examined individual docking poses. The
highest ranked binding mode (Figure 1C, top-1 binding mode) was unlikely to reflect thenatural binding mode of the antibody, since the constant region of Fab was involved in the
HA binding and the binding mode could cause a steric clash between the HA head domain
and F11 Fc region (Figure S2A). In contrast, only the variable domains of F11 participatedin the HA binding and no steric crush was predicted to occur in the binding mode with thesecond highest binding affinity (Figure 1E and Figure S2B, top-2 binding mode). The top-2
binding mode featured the crosslinking of two HA monomers by F11 Fab variable regions(Figure 1E). The top-2-like binding modes involving HA crosslinking were most frequently
seen among the 100 docking poses (Figure S2C). These results suggest that the F11 Fabfragment has physicochemical properties that crosslink two HA monomers by binding to
the stalk domain of the glycosylated HA trimer ectodomain.
3.2. MD Simulation of Glycosylated HA Trimer Docked to F11 Fab Fragment
To assess the stability of the HA-F11 Fab complex, we ran an MD simulation using the
top-2 binding mode (Figure 2). The RMSDs sharply increased after the onset of the MD
simulations, reaching a near plateau after 100 ns, but with greater levels of fluctuations ascompared with those of the unliganded HA trimer (Figures 1A and 2A). This structuralfluctuation mainly reflected that of the F11 Fab linker region between the variable region(VR) and constant region (CR), and fluctuations of the VR for HA binding were minimal, if
any (Figure 2B). Consequently, no major shifting in the binding site was detected before
and after the MD simulation (Figure 2C). These results suggest that the top-2 binding modeis thermodynamically stable under solution conditions.

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Figure 1. Molecular modeling of the complex of glycosylated HA trimer and F11 Fab fragment. A three-dimensional modelof the unliganded, glycosylated HA trimer ectodomain of A/Narita/1/2009 (H1N1)pdm09 virus [19] was constructed by
the homology modeling method using the Molecular Operating Environment (MOE) (Chemical Computing Group Inc.,
Montreal, QC, Canada), glycosylated using tools in GLYCAM-Web [21], and subjected to MD simulation using modules in
the Amber 16 program package [23]. The HA trimer model in the equilibrium state under solution conditions was used
for the docking simulations of the F11 Fab fragment using the Dock application of MOE [29,30]. (A) Root mean squaredeviation (RMSD) [23] between the structure of the initial HA structure and those at given time points of the MD simulationof glycosylated HA trimers calculated using the cpptraj module in AmberTools 16 as described previously [22]. (B) Side
view of the HA trimer model at 400 ns of MD simulation. (C) Distribution of binding energies among the top 100 binding
poses generated using the Dock application of MOE [29,30]. (D) Superposition of 100 docking poses. The purple region
indicates the HA trimer. Green regions indicate the hydrophobic groove [50] in the HA trimer [53,54]. Grey regions indicate
F11 Fab variable regions of the top 100 docking poses. Glycans attached on the HA trimer are not shown here in order tohighlight the binding mode of the F11 Fab variable region. (E) The second highest ranked binding mode of F11 Fab (top-2
binding mode). Cyan branches indicate the high-mannose oligosaccharide Man5 GlcNAc2 .

3.3. Characterization of Molecular Interactions between the HA Trimer Ectodomain and F11
Fab Fragment
The above results suggest that the top-2 complex represents a biologically, physically,
and thermodynamically favorable binding mode with the highest binding affinity to the
HA ectodomain. Therefore, we further examined details of the molecular interactions using
the top-2 structures derived from MD simulation. HA-F11Fab complexes during 180 to
200 ns of MD simulations were used to identify noncovalent interactions between the twomolecules (Figure 3). The binding interface of HA during MD simulations consisted of
16 amino acid residues near the hydrophobic groove [50], the proteolytic cleavage site,
and the fusion peptide (Figure 3A, left panel). The binding surface of F11 during MD
simulations consisted of 20 amino acid residues in the complementarity determining region(CDR) of the heavy and light chains (Figure 3A, right panel). Interactions at individual
contact sites could be classified into three groups (Figure 3B): (1) those between threeglycans in the HA stalk domain and CDR of F11 heavy/light chains; (2) those between
HA and the F11 light chain; and (3) those between HA and the F11 heavy chain. Closely
overlapping hydrophobic regions existed around I341 in the HA proteolytic cleavage loop
and F11 I30 in the light chain CDR1 region (Figure 3C). These data suggest that the F11 Fabfragment could bind to trimeric HA via long-range interactions near the functional sites ofthe HA stalk.

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Figure 2. MD simulation of glycosylated HA trimer docked to the F11 Fab fragment. The top-2 complex was subjected to
MD simulation at 1 atm and at 310 K in 0.15 M NaCl for 200 ns. (A) RMSDs between the structure of the initial HA-F11
Fab complex and those at the given time points of the MD simulation are shown. (B) RMSDs of structural units of the
F11 Fab fragments during the MD simulations. The entire region (Full), variable region (VR), constant region (CR), andunstructured linker region between VR and CR (linker) are shown for the heavy chain (left panels) and light chain (rightpanels). (C) Superposition of the top-2 complex structures before and after 200 ns of MD simulations.

Figure 3. Characterization of molecular interactions between the HA trimer ectodomain and F11 Fab fragment. HA-F11
complex structures collected at every 1 ns during 180 to 200 ns after MD simulations of the top-2 binding mode were used
to search for the sites supporting molecular interactions between HA and the F11 antibody using the Contact Analysis
application of MOE. (A) Visualization of contact sites in the HA trimer (left, wine red residues) and F11 Fab variable region(right, light green and blue green). (B) Interaction networks between HA and F11 Fab variable regions. Non-covalentinteractions detected during MD simulation were visualized using Cytoscape software version 3.8.2 [34] as described
previously [33]. (C) Distribution of hydrophobic patches in the binding interface of HA and F11. Hydrophobic patches witha minimum area of 50 Å2 for protein-protein interactions were estimated using the Protein Patch Analyzer tool in MOE as
described previously [31]. (D) Variation of individual amino acid residues forming noncovalent interactions during MD
simulation. Amino acid sequences of the HA protein of the influenza A virus H1 (n = 36,564) and H3 (n = 53,989) wereobtained from the GISAID database [42]. Shannon entropy [43] was calculated for individual contact sites detected during
MD simulation as described previously [31,41].

HA stalk amino acid residues constituting the contact sites were basically highly
conserved within influenza A/H1 viruses except for a few sites (Figure 3D, left panel),Viruses 2021, 13, 1733

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whereas they were markedly different between the influenza A/H1 and A/H3 viruses(Figure 3D, right panel). These results suggest that the F11 antibody is primarily effective
on A/H1 viruses but not on A/H3 viruses. This prediction is consistent with the previous
finding that the F11 IgG antibody effectively blocked infections by A/H1 viruses, but not
A/H5 viruses [18].
3.4. Effects of F11 Fab Binding on the Structural Fluctuations of the HA Ectodomain
Structural fluctuations of biological macromolecules in solution play key roles inmolecular interactions [55–58] and thus in the biological phenotypes of viruses [18,31–33].
To gain insights into the structural impact of F11 binding to HA, we compared fluctuationsof the HA ectodomain between the free and F11-bound forms using root mean square fluctuation (RMSF), a quantitative indicator of atomic fluctuation [23] (Figure 4). As expectedfrom previous reports [18,31,32], structural fluctuations were often seen in the loops ofthe ligand-free HA glycoprotein (Figure 4A, free HA). Notably, the proteolytic cleavage
loop in the stalk domain, which is the target of cellular proteases [59–62], was found tofluctuate most heavily in the HA ectodomain under solution conditions. In contrast to
the marked impacts on the HA cleavage site, F11 binding to the HA ectodomain did notaffect fluctuations in the regions for the HA receptor binding in the head domain [63,64](Figure 4A, 130 loop, 190 helix, and 220 loop). Interestingly, F11 binding to HA markedly
reduced the structural fluctuations around the cleavage loop, as indicated by the marked
reduction in the RMSF values (Figure 4A, F11-bound HA). The suppression was detectedin the residue at position 327 (T327), residues in the cleavage loop, and residues in thefusion peptide for membrane fusion [50] (Figure 4A). It should be noted that these regions
were not direct contact sites of F11, except for the T327 neighboring of an F11 contact
residue K328 (Figures 3A and 4B). Instead, the regions that were influenced by the F11binding were basically assembled to lie within the F11 binding sites (Figure 4B). These
results suggest that F11 binding could structurally and physically alter the conformational
rearrangement of the HA ectodomain.
3.5. In Silico Site-Directed Mutagenesis
To assess the relative contributions of individual contact residues of F11 to the binding
affinity of F11 Fab to the HA stalk domain, we conducted in silico site-directed mutagenesisof the contact sites. The F11 Fab residues that were capable of interacting noncovalently
with HA (Figure 3A) and the surrounding residues were individually exchanged with
19 nonself residues, and their effects on F11 stability and activity were analyzed as described
previously [44–46] (Figure 5). The single substitutions tested had little effect on the stabilityof the F11 Fab fragment (Figure 5A), as expected from the fact that the substitutions wereintroduced in the variable region of the antibody. In contrast to the stability, the F11
substitutions had some impact on the calculated binding affinity of Fab to HA, althoughthese effects were minor, mostly consisting of changes in the free energy of binding (∆∆G)
within 5 kcal/mole or less (Figure 5B).
We chose eight single substitutions for further analyses. These F11 substitutions
presumably had a different influence on the binding affinity to the HA ectodomain without
affecting the F11 stability (Figure 5C,D). Single substitutions at positions 64 and 65 (Q64W,
G65F, and G65Q) in the F11 heavy chain, which noncovalently interacted with HA residues
near the upper part of the hydrophobic groove during MD simulation (Figure 3A,B), werepredicted to augment the binding affinity (Figure 5D). G65P substitution was predicted
to cause no major changes in the binding affinity, suggesting that the types of side chainat position 65 are important to maintain the binding affinity. Substitutions at position
93 in the F11 light chain, which exhibited no noncovalent interactions with HA during MD
simulation, somehow augmented the binding affinity. In contrast to these findings, single
substitutions at position 49 in the F11 light chain, which noncovalently interacted withglycan on asparagine at position 40 (N40) near the cleavage site of HA (Figure 3A,B), werepredicted to attenuate the binding affinity (Figure 5D).

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Figure 4. Effects of F11 Fab binding on the structural fluctuations of the HA ectodomain. Free and F11 Fab-bound trimeric
HA ectodomains were subjected to MD simulations for 200 ns using the Amber 16 program package [23]. RMSF values of
the Cα atoms of individual HA residues were calculated using 10,000 snapshots during 180 to 200 ns of MD simulations as
described previously [18,31,32]. (A) Distributions of RMSF in HA. Numbers on the horizontal axes indicate positions in the HAof influenza A/Narita/1/2009 (H1N1)pdm09 [19]. Magenta arrows indicate the residues contacting F11 Fab during 180 to
200 ns of MD simulations of the top-2 binding mode. The gray shadows indicate locations of HA residues constituting thehydrophobic groove. Regions involved in sialic acid binding [63,64], such as the 130 loop, 190 helix, and 220 loop, are mergedwith black bars. (B) Structure around the proteolytic cleavage loop. The cleavage loop, fusion peptide [64], and hydrophobic
groove [53,54] are shown in cyan, purple, and orange, respectively. A red arrowhead indicates the cleavage site. Residues that
noncovalently interacted with the F11 Fab fragment during MD simulation are marked with a wine red color.

Figure 5. In silico site-directed mutagenesis. F11 Fab residues capable of noncovalently interacting with HA (Figure 3A)
and the surrounding residues were individually exchanged for 19 nonself residues, and changes in the stability and binding
affinity of F11 Fab were calculated using the F11/HA complex structure obtained at 200 ns of MD simulations as described
previously [44–46] using the Protein Design application of MOE. (A,C) Effects on the stability of the F11 Fab fragment.
(B,D) Effects on the binding affinity of the F11 Fab fragment to the HA ectodomain. (C,D) Results on F11 mutants used forthe experimental mutagenesis.

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3.6. Experimental Mutagenesis
To assess the predictive validity of the in silico mutagenesis, we conducted an experimental mutagenesis study (Figure 6). We assumed that an increase in binding affinity
by a given substitution might increase the neutralization activity of F11 against not only
the original A/Narita/1/2009 (H1N1)pdm09 virus but also its variants. In this regard, a
study on neutralization-resistant viruses might have virological significance. Therefore,
we included two A/Narita/1/2009 (H1N1)pdm09 variants here, C1 and G6, that possess
single mutations in the HA stalk domain and exhibit different levels of reduced sensitivity
to the F11 IgG antibody: C1 has a highly resistant phenotype and G6 has a weakly resistantphenotype [18]. The C1 virus possessed a T333K substitution that has been reported to be
involved in resistance to a broad range of anti-HA stalk antibodies in infection of MDCK
cells by the A/Netherlands/602/2009 (H1N1)pdm09 virus [10].

Figure 6. Experimental mutagenesis. Single amino acid substitutions were introduced into the Fab fragment of the F11
IgG1 antibody according to the information in Figure 5C,D. Affinity-purified soluble IgG1 antibodies were used to assess
neutralization activity using humanized MDCK cells and 100 TCID50 of influenza virus as described previously [48,49].
Neutralization activity was defined as the reciprocal of the lowest concentration (µg/mL) of antibody at which A630 was>X as calculated using the formula X = (A cell − A virus)/2. (A) SDS PAGE of the purified F11 IgG fractions used for theneutralization assay. (B) Neutralization activity against the F11-sensitive (H1N1)pdm09 virus [18] (left panel), F11-resistant(H1N1)pdm09 variant C1 [18] (middle panel), and F11-weakly resistant (H1N1)pdm09 variant G6 [18] (right panel). NT
activity is presented in the scatter plots as the geometric mean, with the geometric standard deviation from four technicalreplicates (except for F11 WT: n = 10). Y-axis values represent neutralizing titers, calculated as 100/minimum concentration(µg/mL) of antibody that neutralized the virus. Dotted lines represent the detection limit (y = 1; 100 µg/mL). ** p < 0.01 and**** p < 0.0001, comparing F11 mutants with F11 WT (Kruskal–Wallis test, followed by Dunn’s multiple comparison test).
For statistical analysis, a provisional minimum NT activity value (y = 0.5; 200 µg/mL) was applied to samples in which NT
activity was below the detection limit.

Purified IgG1 fractions of F11-derived mutants were prepared as described previously [18] and used for a neutralization assay with humanized MDCK cells [48,49]. We

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confirmed successful production and purification of F11 IgG1 variant antibodies by SDSPAGE (Figure 6A), as expected from in silico data on the stability of F11 Fab mutants(Figure 5C). Individual F11 mutations appeared to have distinct effects on the neutralization activity against the A/Narita/1/2009 (H1N1)pdm09 virus (Figure 6B, left panel).
G65F in the F11 heavy chain, which was predicted to achieve the greatest increase in thebinding affinity to HA (Figure 5D), consistently increased the neutralization activity of F11
by about two-fold, although statistical significance was not obtained (Figure 6B, G65F inthe left panel). More remarkably, single substitutions at position 49 in the F11 light chain,
which was predicted to moderately reduce the F11 binding affinity to HA (Figure 5D),
consistently reduced F11 neutralization activity (Figure 6B, Y49G/P in the left panel). No
statistically significant changes were detected with the other F11 mutants.
As expected from the previous study [18], the purified F11 IgG1 fraction had little
neutralization activity against the F11-resistant C1 virus (Figure 6B, WT in the middle
panel). Most of the F11 single substitutions also had little activity. Notably, however, G65F
substitution was found to confer an important new phenotype on F11 IgG1: neutralization
activity against the C1 virus (Figure 6B, G65F in the middle panel. The G65F substitution
also had positive effects on activity against the G6 virus, as it did against the original
virus (Figure 6B, G65F in the left and right panels). Here again, a marked reduction
in neutralization activity was detected with F11 possessing Y49G/P substitutions. No
statistically significant changes were detected with the other F11 mutants. Thus, the insilico data on the binding affinity, i.e., F11 substitutions exhibiting the greatest positive andnegative effects on in silico binding affinity, were consistent with the positive and negative
effects on neutralization activity.
4. Discussion
In this study, we characterized the molecular interactions between an anti-HA stalk
antibody F11 Fab fragment and the trimeric form of the influenza A(H1N1)pdm09 HA
glycoprotein. We then used this information to produce F11 variants with new phenotypes
by single substitutions in the F11 variable region. Thus far, there has been only a single
report on the structure-based creation of new antibodies against influenza viruses [65].
That earlier study presented a successful case of modification of an anti-neuraminidase
antibody. To our knowledge, however, the present study is the first report to describe
the creation of an anti-HA stalk antibody possessing neutralization activity against aneutralization-resistant virus.
In this study, we have constructed and characterized 100 complex models of an HA
ectodomain docked to an F11 Fab fragment using homology modeling, MD simulation, anddocking simulation to understand the structural basis of the actions and modifications of
this antibody. Our in silico study with the 100 models disclosed that the F11 Fab fragment
has physicochemical features that crosslink two HA monomers by binding to a region
near the proteolytic cleavage site of the stalk domain (Figure 1). This unique binding
mode is represented by the top-2 docking pose with the lowest free energy of binding andstability during MD simulation (Figure 2). Variation in the HA residues contacting the F11
Fab in this binding mode explains the neutralization specificity of the F11 antibody [18](Figure 3) well. Moreover, the F11 Fab binding to HA was predicted to have little effect on
the structural dynamics of the surfaces on the HA head domain for the sialic acid binding(Figure 4), consistent with our previous observation that the F11 antibody did not suppress
the hemagglutination inhibition activity of HA [18]. Finally, F11 derivatives exhibiting
positive and negative effects on neutralization activity were effectively obtained with the
top-2 binding mode (Figures 5 and 6). Collectively, these results strongly suggest that thebinding mode proposed in this study represents a physicochemically, thermodynamically,and virologically reasonable mode for the F11 Fab fragment. To date, similar binding
modes involving the crosslinking of two HA monomers have often been reported with
anti-HA stalk antibodies [9,12–14,51,52]. Thus, the crosslinking is likely to represent afavorable binding mode for the anti-HA stalk antibodies in general.

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Results of our present in silico studies suggest that there are at least two types ofstructural impacts for the F11-mediated neutralization of influenza viruses. First, theF11 binding to HA may disturb the cleavage of HA that is a prerequisite for the ordered
conformational rearrangement to establish virus infection of the cells [11,60]. Our study
suggests that F11 Fab can bind to a region near the proteolytic cleavage site of the stalk
domain (Figure 3) and suppress the dynamics of structural units, such as the cleavage loop,
proteolytic cleavage site, and fusion peptide (Figure 4). Because the structural dynamics
of the molecular interaction surface is critical for the molecular interactions and thus
the function of biomolecules [18,31–33,55–58], it is possible that F11 binding not only
sterically but also physically inhibits the proteolysis of HA by cellular proteases. In thisregard, it is worth noting that proteolysis of HA is suggested to occur with proteases inhuman respiratory epithelial cells [59,66]. Such proteolysis can occur with HA on thevirion by the cell surface human airway trypsin-like protease [59,66] prior to endocytosis.
Therefore, F11 binding to HA on the incoming virion outside of the cells may be able todisturb the proteolytic cleavage during virus entry and consequently block initiation of theconformational rearrangement of HA in the cells.
Alternatively, the F11 binding to HA may inhibit pH-dependent conformationalchanges in the endosomes that are required for viral membrane fusion and genome uncoating in the cells [11,50,61,67]. Our in silico study suggests that the F11 Fab fragment
can crosslink two HA monomers by binding to a conserved region near the proteolytic
cleavage site of the stalk domain (Figure 3). This binding mode should sterically preventthe conformational changes in endosomes, such as exposure of the fusion peptide, andthus block subsequent processes, such as membrane fusion and genome uncoating, in thecase that F11 maintained its binding ability to HA under acidic conditions. Further study
is necessary to address each of these possibilities.
Next, we attempted to modify F11 based on the structural information related to the
F11 Fab-trimeric HA ectodomain interactions. Structure-guided mutagenesis of the F11
antibody led us to identification of a residue 49 in the F11 light chain as a critical site for theF11-mediated neutralization (Figures 5 and 6). Single amino acid substitutions at this site
almost completely abolished the neutralization activity of F11. Reduction in the binding
affinity was constantly detected for each of the complexes during the MD simulations,suggesting the affinity reduction could occur in solution (Figure S3). However, it is hard
to explain the complete abolishment of F11 neutralization activity by affinity reduction
alone because the levels of reduction were rather small, and because considerable affinities
remained during the MD simulation (Figure S3). Therefore, it is likely that additional factors
in addition to the affinity reduction are involved in the dysfunction of F11 possessing thesingle substitutions at position 49. In this regard, it should be noted that the F11 residue at
position 49 presumably interacts noncovalently with an HA glycan moiety on asparagine atposition 40 (N40) near the HA cleavage site by forming a hydrogen bond, and that a single
substitution at position 49 of F11 could result in the loss of this interaction (Figure S3).
These results suggest that the free glycan moiety on N40 is critical for virus infection ofcells. Nonetheless, the function of this glycan in viral protection against the anti-HA stalk
antibody [14,68], as well as its function in virus infection and replication have not beenreported thus far. Further study on the function of this particular glycan near the HA
cleavage site may provide new insights into the mechanisms of influenza virus infection
and replication. It is important to study such resistance mutations and develop methods toovercome the resistance, because antibodies targeting the stalk domain can be a powerfultool to combat infections of influenza viruses [6–8].
We previously isolated two A(H1N1)pdm09 variants, the C1 and G6 viruses, thatexhibited potent and weak resistance to F11, respectively [18]. Interestingly, a recent
study has shown that T333K, a single substitution detected in the C1 virus, is involved inresistance to a broad range of anti-HA stalk antibodies [9,10], indicating that the residue isa key player for the anti-HA stalk antibody resistance. Curiously, a virus possessing theT333K substitution still retained an ability to bind to the anti-HA stalk antibodies FI6 [10].

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The results indicate that the T333K substitution functions in the resistance by mechanismsthat are not involved in the prevention of antibody binding. One such mechanism maybe the T333K-induced restoration of dysregulated structural dynamics of HA caused by
antibody binding. In this study, we have suggested by MD simulation that F11 antibody
binding to the trimeric HA induces dysregulation of structural dynamics in the fusion loop
and its flanking regions (Figure 4). This structural defect may be restored by the T333K
substitution, so that proteolytic cleavage proceeds to initiate conformational changes for
infection. Further studies involving MD simulation will help to address this issue.
Our structure-guided mutagenesis of the F11 antibody in this study led to identificationof an F11 derivative that suppresses infections by the C1 and G6 viruses (Figures 5 and 6).
Importantly, G65F single substitution in the F11 heavy chain was sufficient to overcome theresistance (Figure 6B). At present, the molecular mechanism underlying this phenomenon
remains unclear. A possible mechanism might be a dysfunction of the HA moieties by
G65F substitution. The F11 residue at position 65 in the heavy chain presumably contacts
the amino acid and glycan residues near the hydrophobic groove (Figure 3A,B). Thusfar, the functions of these HA moieties in virus infection and replication have not beenreported. Interestingly, the G65F substitution was suggested to induce drastic changes in
the interaction networks around R62, K319, and M403 (Figure S3B), which may suppress the
structural dynamics around these residues. Further structure–functional studies on theseparticular amino acids and glycan residues may provide new insights into the mechanisms
of influenza virus infection and replication for antibody modification.
In conclusion, we here proposed a physicochemically, thermodynamically, and virologically reasonable model of the anti-HA stalk antibody F11 and trimeric HA ectodomain.
The model involves crosslinking of two HA monomers and has provided new clues forunderstanding the structural mechanisms of virus neutralization by the F11 anti-HA stalk
antibody. Moreover, the model was found to be useful to identify a critical site for theF11-mediated neutralization, as well as to generate anti-HA stalk antibodies that acquired
the ability to suppress infections of F11-resistant viruses. Since the anti-HA stalk antibody
is a potential tool to combat the influenza virus [6–8], it would be important to understandthe mechanisms that underlie the neutralization of neutralization-resistant viruses using
the anti-HA stalk antibody derivative obtained in this study.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/v13091733/s1: Figure S1: Amino acid sequences of the F11 Fab fragment and HA proteinof the influenza A/Narita/1/2009 (H1N1)pdm09 strain; Figure S2: Binding modes in the docking
simulations of the F11 Fab fragment and glycosylated HA trimer of influenza A/Narita/1/2009(H1N1)pdm09; Figure S3: Effects of the F11 Y49P and G65F substitutions on molecular interactions.
Author Contributions: Conceptualization, O.K. and S.S.; methodology, O.K., Y.S., A.A. and S.S.;
software, O.K. and M.Y.; validation, O.K., Y.S., A.A., M.Y., K.S., K.T. and S.S.; formal analysis, O.K.,
Y.S., A.U., T.H. and S.S.; investigation, O.K., Y.S., A.U., T.H. and S.S.; resources, O.K., Y.S., K.S., K.T.,
T.S., H.H. and S.S.; data curation, O.K., Y.S. and S.S.; writing—original draft preparation, O.K., S.S.
and H.S.; writing—review and editing, H.S., O.K., Y.S., S.S., A.A., A.U., T.H., K.S., K.T., M.Y., T.S. and
H.H.; visualization, O.K. and S.S.; supervision, T.S., H.H. and H.S.; project administration, O.K. and
S.S.; funding acquisition, O.K., S.S., A.A. and H.S. All authors have read and agreed to the publishedversion of the manuscript.
Funding: This work was supported in part by grants from the Japan Agency for Medical Researchand Development, AMED (Research Program on Emerging and Re-emerging Infectious Diseases:
JP20fk0108107 and JP21fk0108107 to OK, AA, and SS, and JP20fk0108118 and JP21fk0108118 to HS).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Please contact the corresponding authors (O.K. and S.S.) for data on
the present study.

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Acknowledgments: The MD simulations were partly carried out with the TSUBAME3.0 supercomputer at the Tokyo Institute of Technology. We thank Hiromi Nakamura and Akiko Sataka for theirtechnical assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the designof the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; orin the decision to publish the results.

Abbreviations
CDR
CR
DMEM
EHT
HA
hCK
MD
MDCK
MEM
MOE
NT
RMSD
RMSF
VR

complementarity determining region
constant region
Dulbecco’s Modified Eagle’s Medium
Extended Huckel Theory
Hemagglutinin
Humanized MDCK
Molecular dynamics
Madin-Darby canine kidney
Minimum essential medium
Molecular Operating Environment
microneutralization
Root mean square deviation
Root mean square fluctuation
variable region

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