4HVH transgenic mice and control wild-type (WT) mice were immunized according to the protocol approved by the Dutch Experimental animal committee DEC Nr EUR 1944. Briefly, mice were injected i.p. five times at 2-week intervals with the influenza virus X-31 hemagglutinin (HA), prepared as described by Ruigrok et al. (17), and dissolved in phosphate-buffered saline (PBS) at pH 7.4 using Stimune adjuvant (Prionics, Switzerland) according to the formulation provided by the supplier. The last injection was without the adjuvant. Four days after the last injection, 4HVH mice were sacrificed and PCs isolated.

A single-cell suspension was prepared from spleens and femurs in 0.5% BSA, 2 mM EDTA in PBS. Cells were counted (Burke chamber), and magnetic cell sorting of CD138⁺ cells was performed using mouse CD138⁺ plasma isolation kit (Miltenyi Biotec GmbH, Germany) according to the manufacturer’s instructions. Basically, this consists of two steps: first a depletion of non-PCs by indirect magnetic labeling of CD49b and CD45R cells with a non-PC depletion cocktail and anti-biotin microBeads followed by magnetic separation using LD columns (Miltenyi Biotec GmbH, Germany), and the next step is a positive selection of PCs by direct labeling with CD138 MicroBeads followed by magnetic separation on a MS column (Miltenyi Biotec GmbH, Germany). PCs eluted from the column were spun down and the pellet resuspended in 400 µl of Ultraspec™ RNA reagent (Biotecx laboratories, Inc., Houston, TX, USA). Total RNA was made according to the manufacturer’s instructions. The RNA was dissolved in 20 µl of dH₂O and 3 µg was used in a 20 µl reaction volume for a first strand cDNA synthesis using SuperScript™ II RT (Invitrogen by Fisher Scientific, USA) according to the instructions using oligo dT priming with oligo (dT) 12-18 primer (Invitrogen by Life Technologies, USA). The transgenic mice contain four different VHs (1-46, 3-11, 3-53, and 3-23), and hence three different leader-specific primers were designed for the 5′-end. All of them contained a PvuII site that is unique in the final expression vectors (pCAG hygro G2 and pCAG hygro G3 containing leader sequence from VH3-23). The following 5′-primers were used:

lib-3-23/53-S: 5′-GTGTCCAGTGTGAGGTGCAGCTG-3′,

For the 3′-end, we used primer HINGEIgG2rv [previously used and described for phage display library (3)] and HINGEIgG3rv: 5′-AATTGTGTGAGCGGCCGCACCAAGTGGGGTTTTGAGCTC.

An additional 3′-end primer was used: lib-IgG2/3-CH3-AS: 5′-CTGACCTGGTTCTTGGTCATCTCCTC.

This primer from the CH3 constant region is common for both G2 and G3. However, in combination with any of the 5′-primers, it amplifies only IgG2. To make sure that all of the VHs were represented in the library, each of the 5′-end primers was used separately in combination with each of the 3′-end primers, and the products were mixed in an equimolar ratio later. PCR was performed with high-fidelity DNA polymerase Phusion™ (New England Biolabs, Inc., USA) using cycling conditions recommended by the manufacturer for the three-step protocol with an annealing temperature of 68°C and 35 cycles in total. PCR products for IgG2 were cut either with PvuII/BstEII or PvuII/BsrGI (unique site in the constant region) if the amplified fragment originated from using the combination with the Lib-IgG2/3-CH3 primer and cloned into PvuII/BstEII or PvuII/BsrGI cut and phosphatase-treated pCAGhygro G2 vector.

PCR products for IgG3 were digested with PvuII/BstEII or PvuII/SacI and cloned into PvuII/BstEII cut and phosphatase-treated pCAGhygro G2 or PvuII/SacI cut and phosphatase-treated pCAGhygro G3 vector.

pCAGhygro G2 and pCAG hygro G3 are depicted in Figure 2. In short, the vector contains an ampicillin resistance gene for bacteria selection and a hygromycin resistance for the eukaryotic cell selection. The HCAb expression is driven by ubiquitous CMV enhancer and chicken beta actin promoter. The human VH 3-23 leader is responsible for secretion of the antibody. Constant regions have a deleted CH1 exon.

All ligations were done overnight at 16°C in 10 µl total volume using 120 ng of vector alone (control) or in combination with 30 ng of insert using T4DNA ligase (Promega, Madison, WI, USA). Ligations were diluted 5× (40 µl of dH₂O into 10 µl ligation), and 1 µl was used to transform 20 µl of electrocompetent MegaX DH10B™ T1^® cells (Invitrogen by Life Technologies, Grand Island, NY, USA) according to the instructions provided by the manufacturer.

Plating was done on 2XTY/Amp agar plates, and 960 individual colonies were picked into 1.5 ml of 2XTY/Amp medium in 96-well format. A total of 960 DNA plasmid preparations were done using NucleoSpin R96 flash (Macherey-Nagel, Germany) designed for rapid manual and automated 96-well DNA preparation of high- and low-copy plasmid and Bac DNA from Escherichia coli. Each DNA was dissolved in 50 µl sterile dH₂O. A total of 200 ng of DNA (estimated to be in10 µl of DNA preparations based on random sample concentration measurements) was used for transfection into HEK 293T cells. The HEK 293T cells were plated into 96-well plates and transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 96-well format. The following day, medium was removed and replaced with DMEM medium (Lonza, Belgium) supplemented with hygromycin (Roche, Diagnostics GmbH, Germany) at a concentration of 200 µg/ml, non-essential amino acids (NEAA, Lonza, Belgium), and 10% fetal calf serum (FCS). On day 4 posttransfection, 100 µl of medium was taken from each well for an antigen-specific enzyme-linked immunosorbent assay (ELISA) and replaced with a fresh medium.

Enzyme-linked immunosorbent assay plates were coated overnight at 4°C with 5 µg/ml of antigen in PBS or PBS only. Blocking was done for 1 h at room temperature (RT) with 1% milk 1% BSA/PBS (W/V). This was done to exclude possible “sticky” binders to plastic or non-specific binders to BSA/milk. Washing steps included 3× PBS/0.05% Tween-20 and 3× PBS. A total of 50 µl of transgenic mouse serum were diluted in PBS (for initial testing of immunized animals), or supernatants from HEK 293T cells were mixed with 50 µl of 2% milk 2% BSA/PBS (W/V) and incubated for 1 h at RT. After washing, the antigen-specific serum/supernatants were detected by incubation with goat antihuman IgG Fc coupled to horse radish peroxidase (HRP; Jackson Immuno Research Laboratories, Inc., USA) diluted 1:5,000 for 1 h at RT, followed by washing steps and incubation with peroxidase substrate BM Blue POD (Roche Diagnostics GmbH, Germany). The reaction was stopped with 1M H₂SO₄, and the absorption was measured at 450 nm (against reference wavelength 690 nm).

For the initial testing of immunized WT and 4HVH transgenic mice sera for the presence of antigen-specific mouse antibodies, polyclonal goat antimouse immunoglobulins, HRP (Dako, Denmark) diluted 1:2,000 was used.

Sequencing of the positive DNA clones was done using primer CAG seq2-s 5′-GCTGGTTATTGTGCTGTCTCATC-3′.

Medium was collected from HEK 293T cells, stably transfected with an HCAb expression vector and grown to confluence. To screen for the clones that expressed the highest affinity HCAb for HA, 200 µl of HEK 293T cell medium from each clone was transferred to a single well of a black 96-well microtiter plate (Greiner Bio-One, Germany). The binding of the HCAbs to influenza HA was studied using the Octet QK (ForteBio, USA). Anti-human IgG-coated tips (ForteBio, USA) were incubated with PBS/0.05% Tween-20 (120 s) to establish baseline signals. To allow the capturing of HCAbs, the tips were then transferred to 200 µl HEK 293T medium containing HCAbs (600 s), HEK 293T medium without HCAbs and PBS/0.05% Tween-20 as a reference. Subsequently, the tips were transferred to 200 µl PBS/0.05% Tween-20 (480 s) to establish the binding levels after the dissociation of non-specific interactions. Next, the loaded tips were transferred to 200 µl 512 nM bromelain-released HA (BHA; 600 s) to allow HA binding to the captured anti-HA HCAbs and to 200 µl PBS/0.05% Tween-20 (1,200 s) to determine the dissociation rate of the HA from the HCAb.

The reference binding signal was subtracted from the binding curves, and the binding data were fitted to a 1:1 binding model using Octet 4.0 software. The HCAb clones that showed binding in initial affinity screen were selected for further analysis.

Full kinetics experiments were performed with purified HCAbs dialyzed against PBS. Anti-human IgG(Fc) sensors were dipped for 180 s in PBS/0.05% Tween 20. HCAb (10 µg/ml) was used for the loading step (300 s, 900 rpm), followed by 180 s in PBS/0.05% Tween 20. The association step with HA ligand in a concentration range from 0 to 512 nM was performed (600 s at 900 rpm), followed by dissociation step of 1,200 s in PBS/005% Tween 20 at 900 rpm. All steps were done in 200 µl volume. The buffer only well (PBS/0.05% Tween 20) was used as a reference well. The reference binding signal was subtracted from the binding curves, and the binding data were fitted to a1:1 binding model using Octet 7.1 software.

Positive clones were further expanded in medium containing 10% FCS. Alternatively, plasmid DNA was linearized with HindIII, stably transfected into HEK293T cells, and individual clones picked for the purpose of selecting the best expressors.

For production purposes, clones were grown in 15 cm Petri dishes in 25 ml of OPTI-MEM^® (1×) + GlutaMAX™ (Gibco by Life technologies, CA, USA) medium. Medium was collected and replaced twice per week. Collected medium was spun down at 1,000 rpm (Eppendorf centrifuge 5810R) for 5 min to remove cell debris, and HCAbs were purified on Protein A agarose Fast Flow 50% (V/V; Sigma, USA). A total of 100 µl of Protein A beads were incubated with 50 ml of medium overnight at 4°C on a rotating wheel. After spinning at 1,000 rpm for 5 min, beads were washed in PBS/0.01% Tween-20 and loaded onto homemade columns (insulin syringe with cotton wool), washed 3× with PBS/0.01% Tween-20, 3× with PBS, and eluted with 3M potassium thiocyanate (KSCN). Eluted HCAbs were dialyzed for 5–6 h using Spectra/Por dialysis membrane MWCO 10000 (Spectrum Laboratories Inc., USA) against 1,000× volume excess of PBS at 4°C. The procedure was repeated three times.

VH domains from HCAbs were cloned without a tag into pET SUMO vector (Champion pET SUMO Expression system, Invitrogen, CA, USA). Production of fusion protein, cleavage, and removal of SUMO and SUMO protease were done according to the manufacturer’s instruction with recommended reagents.

VH domains in fusion with SUMO were purified from inclusion bodies starting with 200 ml BL21 transfected cells after 4 h of induction (1 mM IPTG). Cells were harvested, pellet frozen overnight, and lysed in 20 ml lysis buffer (50 mM KPO₄, pH 7.8; 400 mM NaCl; 10 mM KCl; 10% glycerol; and 0.5% Triton X-100). After sonication 20 × 20 s ampl 12 and spinning for 15 min at 4,000 rpm at 4°C, pellet was taken in 1 ml PBS with addition of 10 µl benzonase for 30 min at RT. After washing in washing buffer (100 mM Tris pH 7.5; 5 mM EDTA; 2 M urea; and 2% Triton X-100), sample was spun for 15 min at 4,000 rpm, washed 2× in 10 ml of 100 mM Tris pH 7.5 and 5 mM EDTA. Pellet was taken into 8 M urea; 10 mM Tris pH 7.5; and 2 mM DTT, rotated for 2 h at RT, spun for 30 min, 15,000 rpm at 4°C, and the supernatant was used in refolding. Base refolding buffer 2 from Pierce refolding kit was used (440 mM 1-arginine; 55 mM Tris pH 8.2; 21 mM NaCl; and 0.88 mM KCl). Protein was diluted to 1 mg/ml; in 8 M urea, 10 mM Tris pH 7.5; and 2 mM DTT. To fold 0.5 mg of protein, we used 9 ml base refolding buffer, 100 µl 0.1 M EDTA, 6.75 mg GSH (reduced glutathione), 2.65 mg of GSSG (oxidized glutathione), and H₂O up to 9.5 ml. Protein was added in 50 µl aliquots, well mixed, and left on ice for at least 1 min after each addition. After overnight incubation at 4°C, sample was dialyzed against PBS buffer.

A total of 500 µg of each HCAbs was concentrated to an end point of 30 µl volume using Centriprep-10 K centrifugal filter device (Merck Millipore Ltd., Ireland) as suggested by the manufacturer. Concentrated samples were diluted 10× in 8M guanidine hydrochloride (Gdn-HCl) and the OD at 280 nm measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Ireland). The same procedure was used for selected VHs expressed in E. coli.

A total of 2 µg of purified and PBS-dialyzed HCAb in 50 µl of PBS was run on a Superdex 200 (3.2/30) column (GE HealthCare Life Sciences, USA) on the FPLC Smart system from Pharmacia. Samples were run in PBS. Bio-Rad’s gel filtration standard as a mixture of molecular weight markers ranging from 1,350 to 670,000 Da was used as a control. The same procedure was done with selected VHs expressed in E. coli run on a Superdex 75 (3.2/30) column (GE HealthCare Life Sciences, USA).

The microtiter plaque reduction assay, as described by Matrosovich et al. (18), was performed using MDCK-SIAT1 cells. Twofold dilutions of the HCAbs were incubated on cell monolayers prior to addition of virus. The neutralization titer is determined as the reciprocal of the dilution of HCAbs, which corresponds to 50% reduction in plaque formation, compared to the virus control.

Hemagglutination inhibition assays were performed according to standard methods, Kendall et al. (19), using 0.75% and 1.0% of Turkey and guinea pig red blood cell suspensions, respectively. Four HA units and twofold dilutions of HCAbs were used in these assays. HI titers are reciprocals of the highest dilution of HCAbs, which inhibited hemagglutination.

A total of 20 or 50 µg of influenza X-31 HA protein, prepared as described by Ruigrok et al. (17), per mouse was injected intraperitoneally in 2 weeks intervals into eight of 4HVH transgenic mice (two transgenic lines, 4HVH-B and 4HVH-C, originating from different founders, having different integration site of the transgene) and two WT mice as a control, using Stimune as an adjuvant. After the third injection, mice were bled, and an ELISA assay was performed on serum, using HA protein-coated plates (5 µg/ml) and anti-human IgG-HRP or anti-mouse IgG-HRP for the detection of HA-specific antibodies. In seven out of eight transgenic mice and two out of two WT mice, HA-specific antibodies were detected (Figure S1 in Supplementary Material). The higher amount of antigen did not lead to a better response, thus we concluded that 20 µg is sufficient for successful immunization. Seven transgenic ELISA positive mice were immunized three or more times with 20 µg of HA.

Four days after the last injection without adjuvant, mice were sacrificed, and CD 138⁺ PCs were isolated from bone marrow and spleen. Total RNA was isolated, and cDNA was synthesized (depicted schematically in Figure 1). Human VDJ domains were amplified using a set of three different 5′-end primers specific for the leader sequences in the transgenic mouse construct in combination with two different 3′-end primers specific for either the human IgG2 or IgG3 hinge (3). Due to the high sequence similarity, the same 5′-end primer was used for amplification of both VH3-23 and VH3-53. All 5′-primers were designed to contain a PvuII restriction site. A PvuII site appears usually at the beginning of the DNA encoding VH regions (third to fourth codons at the amino acid level) and rarely occurs anywhere else in human VHs. Amplified PCR products were cut either with PvuII/SacI or with PvuII/BstEII restriction enzymes. The SacI site is a unique site at the beginning of the IgG3 hinge, while the BstEII site in frame 4 is unique in most of the VHs and is commonly used for constructing phage libraries (21, 22).

The VDJ fragments were cloned into bacteria using either pCAGhygro G2 or pCAGhygro G3 expression vectors, containing the ubiquitously expressed chicken β-actin promoter, the leader sequence from human VH3-23, and the constant region of human IgG2 or IgG3 (Figure 2). The ligated cDNA was transfected into electrocompetent E. coli cells, totaling 1 part out of almost 700,000 of the available RNA in the mice (PCs were isolated from half of the total number of cells, 1/4 of total RNA was made into cDNA, 1/30 of the gel purified PCR amplified cDNA was used in the ligations; 1/50 of the ligation was transformed, 1/32 of the transformed bacteria was plated, and 0.57 of total colonies counted were picked from those plates). A total of 960 colonies were picked from both the G2 and G3 library into 96-well plates filled with 2XTY medium. The resulting recombinant bacteria were grown overnight, and plasmid DNA was prepared in the same 96-well format. In parallel, HEK 293T cells were also grown in a 96-well format and transfected with the plasmid DNA maintaining the same 96-well format. A 96-well HA-specific ELISA screen was performed with supernatants from each well 4 days after transfection. This yielded 66 positive supernatants/clones. The corresponding cDNAs were sequenced (Figure 3), which showed that two out of the four available variable segments were used preferentially (VH3-11 and VH3-23); the VDJ domains contained somatic hypermutations; J4 was predominantly used but J5 and J6 were also found; both IgG2 and IgG3 antibodies were present, confirming that class switching occurred in the transgenic mouse. Most prevalent are IgG2 HCAbs, and it is not surprising taking into account that the IgG2 constant region is the most proximal in the transgenic construct and thus the first to recombine. Out of 66 sequences, 45 were unique and these account for 33 different DJ regions. Based on the different CDR3 regions, the HCAbs were classified into 13 distinct groups represented in different colors (Figure 3). Positive clones were cultured further, and supernatants were collected for affinity screening, using the same HA antigen preparation. Twenty four clones showed significant binding. A selected number of clones (based on sequence diversity and affinity) were produced in serum free medium. The antibodies with low affinities were not tested at all in a functional assay (see below). Four groups represented by 7F2 (Figure 3, blue), 2F4 (green), 1F3 (red), and 3A8 (yellow) were left. Of these, 1F3 and 2F4 appear to bind the same epitope(s), while 3A8 and 7F2 appear to bind a different but overlapping epitope(s).

Heavy-chain-only antibodies from medium were purified on protein A, eluted in 3M KSCN, and dialyzed against PBS. The average yield was estimated to be 2–4 µg/ml of medium. On SDS-PAGE gel, HCAbs are of expected size for a monomer (~40–45 kDa) under reducing and of a dimer (~80–90 kDa) under non-reducing conditions (Figure 4A). Size differences originate from different sizes and compositions of VH regions and different sizes (longer hinge) of IgG3 HCAbs, accounting for >5 kDa difference per monomer in comparison to IgG2 HCAbs. The profiles on size chromatography (smart columns) showed a single peak of the expected size; the peaks of the IgG3 HCAbs, 3A11, and 3B10 were of higher molecular weight than IgG2 HCAbs (Figure 4B). Solubility in PBS was tested with 500 µg of each HCAb by concentrating it to ~30 µl of final volume. The selected HCAb differed with concentrations up to 15.4 µg/µl for 1F1, the equivalent of 30 mg/ml of a normal H2L2 antibody (Figure 4C). Further concentration of the very soluble antibodies was not tested. Two of the VHDJ segments (3A8 and 2F4) were also expressed in bacteria and shown to be soluble in at least 5 mg/ml, which is also equivalent to 30 mg/ml of a normal H2L2 antibody (Figure S2 in Supplementary Material). Binding affinities of the HCAb were determined on an Octet instrument with purified antibodies. A BHA preparation (23) that lacks a transmembrane anchor was used to prevent rosette formation and aggregation of antigen. The majority of KD values (9 of 17) for HCAbs were in the 10⁻⁹ molar range, 7 were in the 10⁻⁸ molar range, while the KD value for the highest affinity 1F3 HCAb was better than 10⁻¹⁰. HCAbs 1G7, 1G11, 2F4, 1F4, 1C12, 4H1, and 3A11 react on Western blots under both non-reducing and reducing conditions recognizing denatured BHA monomer and the BHA1 chain, respectively. 7F2 reacts with BHA only under non-reducing conditions, while 1F3 preferentially recognizes BHA1 under reducing conditions (data not shown).

The HA HCAbs were first evaluated for their ability to neutralize X.31 (H3N2) influenza virus in a plaque reduction assay. Neutralization is reported as the reciprocal of the highest dilution of the antibodies corresponding to 50% plaque reduction compared to the virus control. A total of 18 of the antibodies tested show visible inhibition, and 50% reduction is observed for four HCAb, 1F3 > 2F4 > 3A8 > 4H1 = 7F2 (Table 1). A modified neutralization experiment was also performed, where antibodies were incubated for 30 min with the virus prior to addition to MDCK-SIAT1 cells. This led to an increased neutralizing effect especially in the case of 2F4 and 1F3 antibodies [2F4 neutralizing at ≥1.4 µg/ml (18nM) and 1F3 at ≥0.9 µg/ml (11.7nM)]. Ferret anti X-31 serum and HC19, an anti-X31 HA mouse monoclonal antibody, were used as positive controls. 3A8 and 7F2 HCAbs also showed an HI performed with Turkey red blood cells (Figure 5) and with guinea pig blood (data not shown), suggesting that 3A8 and 7F2 neutralize infectivity by obstructing the binding of virus to the host cell.