JVI Accepts, published online ahead of print on 14 May 2014 J. Virol. doi:10.1128/JVI.03178-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Single domain antibodies targeting neuraminidase protect against an H5N1 influenza
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virus challenge
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Francisco Miguel Cardoso1,2 , Lorena Itatí Ibañez1,2$, Silvie Van den Hoecke1,2, Sarah De
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Baets1,2, Anouk Smet1,2, Kenny Roose1,2, Bert Schepens1,2, Francis J. Descamps1,2#, Walter
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Fiers1,2, Serge Muyldermans3,6, Ann Depicker4,5, Xavier Saelens1,2
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Belgium
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Brussels, Belgium
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VIB Inflammation Research Center, Technologiepark 927, Ghent, Belgium Department for Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent,
Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussels, Belgium Department of Plant Systems Biology, VIB, Technologiepark 927, Ghent, Belgium Department of Biotechnology and Bioinformatics, Technologiepark 927, Ghent, Belgium Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050
Current address: ICT-Milstein – CONICET, Ciudad Autónoma de Buenos Aires, Argentina Current address: Ablynx NV, Ghent, Belgium
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Address correspondence to: Ann Depicker, [email protected], or Xavier Saelens,
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[email protected]
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Running Title: NA single domain antibodies protect against H5N1
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Word Count for abstract: 220
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Word Count for text: 9790
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ABSTRACT
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Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We
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isolated a set of H5N1 NA-specific single domain antibodies (N1-VHHm) and evaluated their in
26
vitro and in vivo antiviral potential. Two of them inhibited NA activity and in vitro replication of
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clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two
28
methods. First we made N1-VHHb by genetically joining two N1-VHHm moieties by a flexible
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linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm
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moiety the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1
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of both bivalent N1-VHHb formats was 30- to 240-fold higher than their monovalent
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counterparts, with IC50 values in the low nanomolar range. Moreover, single-dose prophylactic
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treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal
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challenge with H5N1 virus, including an Oseltamivir-resistant H5N1 variant. Surprisingly, also an
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N1-VHH-Fc fusion without in vitro NA inhibitory or antiviral activity protected mice against H5N1
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challenge. Virus escape selection experiments indicated that one amino acid residue close to
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the catalytic site is required for N1-VHHm binding. We conclude that single domain antibodies
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directed against influenza NA protect against H5N1 virus infection, and when engineered with a
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conventional Fc domain, they can do so in the absence of detectable NA inhibitory activity.
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IMPORTANCE
41
Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of bird flu caused by these
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viruses occur in many parts of the world, are associated with tremendous economic loss and
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these viruses can cause very severe disease in human. In such cases, small molecule inhibitors
44
of the viral neuraminidase are among the few treatment options for patients. However treatment
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with such drugs often results in the emergence of resistant viruses. Here we show that single
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domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro
3
47
and can protect laboratory mice against challenge with an H5N1 virus, including an oseltamivir-
48
resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain, can protect
49
in vivo even in the absence of NA inhibitory activity. Thus, NA of influenza virus can be
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effectively targeted by single domain antibody fragments, which are amenable to further
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engineering.
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INTRODUCTION
53
Zoonotic influenza A virus infections are a persistent threat due to their pandemic potential. In
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particular, highly pathogenic avian influenza viruses (HPAIV) of the H5N1, H7N1 and H7N7
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subtypes occasionally cross the species barrier between domesticated birds and human. These
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viruses could become transmissible between humans through reassortment with circulating
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swine or human influenza viruses or by gradually accumulating mutations (1, 2). In the last
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decade, zoonotic outbreaks had a major effect on public health. HPAIV H5N1 (3), the swine flu
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(H1N1) outbreak in 2009 (4), and more recently, human infections with H7N9 in South Asia (5)
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illustrate our poor preparedness for pandemic influenza (6). HPAIV H5N1 infection in humans
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has a confirmed case fatality rate of approximately 60%. The high pathogenicity of HPAIV H5N1
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in humans can be attributed to a high replication rate and a broad cellular tropism that can lead
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to systemic virus spread. In addition, deregulated induction of proinflammatory cytokines and
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chemokines (“cytokine storm”) is associated with severe HPAIV H5N1 infections, and can result
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in a disproportionate immunological response (7).
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Influenza neuraminidase (NA) is a homotetrameric type II membrane glycoprotein with sialidase
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activity. The NA catalytic site is located at the top of each monomer, opposite to the tetramer
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interface. NA plays an essential role in the spread of influenza viruses by cleaving sialic acids
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from the host cell receptors and from virions. NA activity also contributes to virus entry by
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cleaving decoy receptors present in mucins that line the layer of respiratory epithelial cells (8).
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Immunologically, NA is the second major humoral antigenic determinant (after HA) and is
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subject to antigenic drift and occasional shift. In addition, experimental influenza vaccines
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supplemented with NA have improved efficacy (9-11). NA is also a co-determinant of influenza
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A virus (IAV) pathogenicity (12-14) and is involved in limiting IAV superinfections and
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reassortment (15). Decreased NA activity has been correlated with H5N1 adaptation to human
5
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airway epithelium (16) and antibodies against NA contribute to protection against H5N1 virus
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challenge in a mouse model (17).
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Hemagglutinin (HA), the other major antigen, and NA cooperate in a tightly controlled way. For
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example, the fitness of mutant IAV virus lacking NA activity can be rescued by selection of HA
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mutants with decreased affinity for receptors containing sialic acid (18-20). These data
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demonstrate the importance of NA during IAV infection, so targeting NA is a rational strategy.
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Indeed, three licensed influenza antivirals, Oseltamivir, Zanamivir and Peramivir, target NA.
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Influenza viruses that are resistant to Oseltamivir frequently emerge in humans. In addition, NA-
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specific antibodies protect mice, and serum anti-NA antibodies are associated with resistance to
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influenza A virus challenge in humans (21).
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Effective prevention and treatment strategies are needed to control H5N1 infections and single-
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domain antibody-based technology have been described as promising antivirals (22). Naturally
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occurring single heavy chain antibodies have been found in sharks and camelids (23, 24).
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Unlike conventional antibodies that are typically composed of a light and a heavy polypeptide
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chain that together determine the epitope specificity, these natural single-chain antibodies are
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composed of heavy chains only, and hence their epitope specificity is confined in a single
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domain (so called VHH, Variable domain of Heavy chain). Single-domain antibody fragments
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derived from camelids are highly specific and robust, easy to produce and flexible, and hence
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they are used as novel therapeutics for many diseases (25, 26). VHHs usually have extended
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antibody loop structures and can interact with cognate antigens with affinities matching the best
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affinities of conventional antibodies (27-29). In addition, VHHs that potently inhibit enzymes
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have been described (30, 31), and they have been used to stabilize membrane proteins in order
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to determine crystal structure (32). The VHH tends to target concave-shaped epitopes, in
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contrast to the planar epitopes that are typically recognized by “classical” light and heavy chain
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antibodies (33). In addition, several examples of VHHs with potent antiviral activity have been
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reported (34-36). We previously reported on an HA-specific H5N1 neutralizing VHH (35) and
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H5N2 neutralizing VHH were reported more recently (37). Moreover, a VHH directed against the
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influenza M2 protein inhibited replication of amantadine-sensitive and amantadine-resistant
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viruses and protected mice against lethal influenza challenge (38). Recently, Harmsen et al.
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described a panel of NA-binding VHHs, some of which displayed cross-subtype NA-binding, but
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the antiviral potential of these VHHs was not evaluated (39).
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Here, we report the isolation and characterization of a set of H5N1 NA-binding VHHs (N1-
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VHHm) of which some have potent NA inhibitory activity and in vitro and in vivo antiviral
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activities. We also describe the production of bivalent in tandem N1-VHHm formats in
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Escherichia coli and N1-VHHm proteins fused to a mouse IgG2a Fc domain in seeds of
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transgenic Arabidopsis plants. Further, we compared the in vitro antiviral activity against H5N1
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clade 1 (Oseltamivir-sensitive and -resistant strains) and clade 2 viruses, and we evaluated the
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N1-VHHm based prophylactic protection of mice against a potentially lethal infection with H5N1
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virus.
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MATERIALS AND METHODS
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Influenza viruses
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H5N1 IAV strains NIBRG-14 and NIBRG-23 were obtained from the UK National Institute for
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Biological Standards and Control, a center of the Health Protection Agency. NIBRG-14 and
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NIBRG-23 are 2:6 reverse genetics–derived reassortants with NA and HA (lacking the polybasic
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cleavage site) segments derived from A/Vietnam/1194/2004 (H5N1) and A\turkey\Turkey\2005
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(H5N1), respectively, and the other six segments from A/PR/8/34 (H1N1) viruses. The H5N1
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H274Y virus described here is a 1:1:6 reverse genetics-derived reassortant, with NA derived
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from A/crested eagle/Belgium/01/2004 (40) carrying the H274Y mutation introduced by site-
124
specific mutagenesis, HA from NIBRG-14, and the remaining six genome segments from
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A/PR/8/34 (41). This virus was rescued by transfection of co-cultured HEK-293T and MDCK
126
cells. The supernatant from these cells was used for end point dilution to obtain a clonal H5N1
127
H274Y virus sample that was subsequently amplified on MDCK cells, pelleted from the cell
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supernatant, and mouse adapted by serial passage in BALB/c mice. All HA segments of these
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H5N1 viruses lack the coding information for the polybasic cleavage site. Following adaptation
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to BALB/c mice, the HA and NA-coding regions of the mouse-adapted NIBRG-14 (NIBRG-14
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ma) and H5N1 H274Y (H5N1 H274Y ma) were sequenced and found to be identical to those of
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the parental viruses. pH1N1 (kindly provided by Dr. Bernard Brochier, Scientific Institute of
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Public Health, Brussels, Belgium) is derived from a clinical isolate of the pH1N1 virus of 2009
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and was adapted to mice by serial passages (42). A/New Caledonia/20/99 was kindly provided
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by Dr. Alan Hay (MRC National Institute for Medical Research, Mill Hill, London, UK). The
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median tissue culture infectious dose (TCID50) and median lethal dose (LD50) of NIBRG-14 ma
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and H5N1 H274Y ma viruses were calculated by the method of Reed and Muench (43). All the
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H5N1 and pH1N1 experiments described above were performed in BSL-2+ rooms.
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139
Baculovirus-based production of recombinant soluble tetrameric neuraminidase
140
A soluble and enzymatically active tetrameric NA (N1rec) derived from A/crested
141
eagle/Belgium/01/2004 (H5N1) was produced by proprietary technology (WO 2002/074795).
142
Briefly, the N1rec expression cassette consisted of the secretion signal of IAV HA (ssHA, 16
143
residues), a tetramerizing variant of the leucine zipper domain of the Saccharomyces cerevisiae
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GCN4 transcription factor (tGCN4, 32 residues) (44, 45), and the extracellular part of NA
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derived from A/crested eagle/Belgium/01/2004 (H5N1) (amino acid residues 53–449) (40) (Fig.
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1A). This N1rec expression cassette was cloned into the pAcMP2 baculovirus transfer vector to
147
produce pAcMP2n1rec. Recombinant baculovirus was generated by co-transfection of
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pAcMP2n1rec and linearized DNA of Autografa californica Nuclear Polyhedrosis Virus (AcNPV)
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(BaculoGold, BD Biosciences) into Sf9 insect cells (derived from Spodoptera frugiperda), using
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the ESCORT IV Transfection Reagent (Sigma-Aldrich) to generate N1rec encoding recombinant
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baculovirus. Sf9 cells infected with AcNPVN1rec were incubated at 28°C in rolling bottles in TC-
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100 medium (Invitrogen), supplemented with 10% FCS and penicillin + streptomycin. Four days
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after infection, the supernatant was harvested and clarified by centrifugation (1 h at 50,000 g).
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Recombinant neuraminidase purification
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All purification steps were carried out at 4°C. Two parts (v:v) of n-butanol were added to three
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parts of cleared AcNPVN1rec-infected Sf9 cell supernatant and the mixture was shaken
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vigorously for 5 min. The aqueous phase, containing soluble N1rec, was separated from the
158
organic phase, diluted 2.5 times in 5 mM KH2PO4 pH 6.6 and passed through a 0.22-µm filter.
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The diluted aqueous phase was subsequently applied to an XK26\70 column (GE Healthcare)
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packed with HA Ultrogel Hydroxyapatite Chromatography Sorbent (Pall), and eluted with a
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gradient of 5-400 mM KH2PO4 pH 6.6 in 4% butanol. Eluted fractions with high NA activity were
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pooled and loaded onto a Blue Sepharose (Sigma-Aldrich) column, equilibrated with 50 mM
9
163
MES pH 6.6 containing 5% glycerol and 8 mM CaCl2. Bound proteins were eluted with 50 mM
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MES pH 6.6 containing 5% glycerol, 8 mM CaCl2 and 1.5 M NaCl. Fractions with NA activity
165
were loaded on a HiLoad 16/60 Superdex 200 (GE Healthcare) gel filtration column pre-
166
equilibrated with 50 mM MES pH 6.6 with 5 % glycerol and 8 mM CaCl2, 150 mM NaCl. Peak
167
fractions containing NA activity were pooled, aliquoted, and stored at -80ºC until used. All the
168
chromatography steps were performed using an Akta purification station (GE Healthcare).
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Neuraminidase activity assays
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NA activity was quantified by measuring the rate of cleavage of the fluorogenic substrate 4-
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MUNANA (2’-4-Methylumbelliferyl-α-D-N-acetylneuraminic acid, sodium salt hydrate (Sigma-
172
Aldrich) into 4-methylumbelliferone. The NA activity reaction was performed in 200 mM NaAc, 2
173
mM CaCl2 with 1 % butanol and 1 mM 4-MUNANA and measured in a kinetic mode, with
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excitation at 365 nm and emission at 450 nm in an Optima Fluorostar. A standard curve of
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increasing concentrations of soluble 4-methylumbelliferone was included to correlate the
176
fluorescence intensity with the molar amount of 4-methylumbelliferone. One NA activity unit is
177
defined as the activity needed to generate 1 nmol of 4-methylumbelliferone per min.
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Fetuin (5 µg/ml; Sigma-Aldrich) was coated on Nunc 96 well plates overnight at 4°C. Excess
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fetuin was washed away with PBS, and IAV dilutions (diluted in PBS with 1 mM CaCl2 and 0.5
180
mM MgCl2), with or without added single domain antibodies, were added and incubated 1 h at
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37°C. The amount of desialylated fetuin was measured by colorimetry to determine binding of
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horseradish peroxidase (HRP) coupled peanut agglutinin (PNA, Sigma-Aldrich). The plates
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were washed three times with PBS + 0.1% Tween 20 and then incubated with 50 µl PNA-HRP
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(2.5 µg/ml in PBS + 0.05% Tween 20) at room temperature. Then the plates were washed three
185
times with PBS, after which 50 µl of TMB substrate (Pharmigen BD) was added and absorbance
186
was measured at 450 nm with a reference at 650 nm. In the Accelerated Viral Inhibition Assay
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(AVINA) (46), IAV dilutions containing the indicated N1-VHHm concentrations were transferred
10
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to a black 96-well plate and 75 μl of 20 μM MUNANA was added and incubated 1 h at 37°C.
189
Then, 100 μl stop solution (0.1 M glycine, pH 10.7, 25% ethanol) was added to each well, and
190
fluorescence was determined. For the AVINA and fetuin substrate assays, we used the
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following amounts of IAV: 7 x 105 pfu of NIBRG-14 ma, 1 x 104 pfu of H5N1 H274Y ma, and 3 x
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104 plaque forming units (pfu) of pH1N1.
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Camelid immunization, phage library construction, and panning
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An alpaca (Vicugna pacos) was injected subcutaneously with 125 µg of N1rec in the presence
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of incomplete Freund’s adjuvant. Injections were once a week for five weeks. On day 39, anti-
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coagulated blood was collected, lymphocytes were isolated using a UNI-SEP density gradient
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separation kit (NOVAmed), and total RNA was extracted. cDNA was prepared using oligo (dT)
198
primers,
199
(GTCCTGGCTGCTCTTCTACAAGG)
200
(GGTACGTGCTGTTGAACTGTTCC). PstI and NotI restriction sites were inserted into the
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amplified sequences using the primers A6E (GATGTGCAGCTGCAGGAGTCTGGRGGAGG)
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and 38 (GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT). The resulting PCR product
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(550 bp) and the pHEN4 vector were digested with PstI and NotI, ligated (47), and transformed
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into electrocompetent TG1 E. coli cells. Transformants were grown in 2xTY medium,
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(supplemented with 100 µg/ml ampicillin and 1% glucose) and infected with helper phage
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M13K07. The resulting VHH phage display library was panned four times using solid-phase
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coated N1rec.
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Production and purification of monovalent neuraminidase-binding VHH (N1-VHHm)
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The VHH coding sequence of the selected N1rec-binding phages (n = 13) was amplified by
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PCR with the primers A6E and 38. The N1rec-binding VHH genes obtained by PCR and the
and
the
VH
and
VHH
genes
were and
amplified
with
primer
primer
CALL001 CALL002
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pHEN6c vector were digested with PstI and BstEII, ligated, and transformed into E. coli WK6
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strain. In the pHEN6c vector, the coding sequence for the N1rec-binding VHH candidates were
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cloned downstream of an N-terminal PelB secretion signal sequence and a carboxyterminal
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hexahistidine tag was introduced (Fig. 1B)(48). For production and purification of soluble VHH,
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pHEN6c-transformed WK6 cells were grown in TB medium supplemented with 100 µg/ml
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ampicillin, 2 mM CaCl2 and 0.1% glucose. After 16 hours, VHH production was induced with 1
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mM of isopropyl β-D-1-thiogalactopyranoside, and the periplasmic fraction was extracted by
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osmotic shock using 0.2 M Tris pH 8.0, 0.5 mM EDTA and 0.5 M sucrose. Periplasmic extracts
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were centrifuged at 8000 rpm and 4°C and the supernatant was applied to a His Select Nickel
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Affinity gel (Sigma-Aldrich). After loading, the column was washed with PBS, and VHHs were
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eluted with 0.5 M imidazole and dialyzed against PBS at 4°C. Monovalent N1rec-binding VHH
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(N1-VHHm) was concentrated with a Vivaspin 5000-kDa cutoff filter (Vivascience). Total protein
223
concentration was determined with the BCA protein Assay kit (Thermo Scientific) and endotoxin
224
levels with a commercial Limulus Amebocyte Lysate-based assay (Toxinsensor Chromogenic
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LAL Endotoxin Assay Kit, Genscript Corp.). We obtained 13 recombinant N1 NA-specific VHH
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proteins, which were named N1-1-VHHm to N1-13-VHHm. An irrelevant monovalent VHH
227
against Arabidopsis thaliana albumin (SA-VHHm) was used as a negative control (49). The N1-
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VHHm sequences were aligned using the MegAlign software (DNAstar) and the VHH CDR3
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electronegativity surface potential was predicted with the open source software ESyPred3D and
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visualized with DeepView (50)(Fig. 1C and 1D).
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ELISA assays
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N1rec or M2e-tGCN4 (44) at 10 µg/ml was used to coat O/N in 96-well Maxisorp (Nunc) plates
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using 100 µl per well. Blocking was performed overnight with 5% skim milk in PBS. Dilution
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series of the 13 N1-VHHm candidates (from 10 µM - 0.1 nM) were incubated for 2 h at 37°C,
12
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and excess VHH was removed by three washes with PBS containing 0.05 % Tween 20. Binding
236
of the N1-VHHm to N1rec tGCN4 or NA globular head moieties was assessed by a mouse anti-
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His-tag antibody fused with horse radish peroxidase (HRP) (1:5000). After three washes with
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PBS, 100 µl of Tetramethylbenzidine (TMB) substrate (Pharmigen BD) was added, and
239
absorbance was measured at 450 nm.
240
N1-VHHm surface plasmon resonance analysis
241
The affinity of monovalent N1-3-VHHm, N1-5-VHHm and N1-7-VHHm for N1rec was
242
determined by surface plasmon resonance (SPR) on a Biacore 3000 platform (GE Healthcare).
243
N1rec antigen was first immobilized (2000 resonance units) into a Series S sensor chip CM5
244
(GE Healthcare) by chemical -NH2 coupling, in 10 mM NaAc pH 4.5. N1rec loaded channels
245
were regenerated with 0.02% SDS. Each monovalent N1-VHHm was diluted in HBS buffer (0.01
246
M HEPES, 0.15 M NaCl, 0.005% Tween 20, pH 6.4) to obtain a gradient ranging from 1.95 to
247
1000 nM. The different N1-VHHs were injected over the N1rec coated chip to record their
248
binding kinetics. The Biacore T00 Evaluation Software was used to calculate the association
249
rate constants (Kon), dissociation rate constants (Koff) and the equilibrium dissociation constants
250
(KD = Koff/Kon).
251
Construction of bacteria-produced bivalent N1-VHH (N1-VHHb)
252
The coding information for N1-3-VHHm and N1-5-VHHm was amplified with primers MH
253
(CATGCCATGGGAGCTTTGGGAGCTTTGGAGCTGGGGGTCTTCGCTGTGGTGCGCTGAGG
254
AGACGGTGACCTGGGT)
255
(CATGCCATGATCCGCGGCCCAGCCGGCCATGGCTGATGTGCAGCTGGTGGAGTCT)
256
introduce an NcoI restriction enzyme site at both ends of the amplified fragment. The MH primer
257
also introduced the hinge sequence of llama γ2c (AHHSEDPSSKAPKAPMA) (51) to separate
258
the two N1-VHHm domains. PCR amplicons of N1-3-VHHm and N1-5-VHHm were cloned into
and
A4short to
13
259
pHEN6c plasmid containing the n1-3-vhh or the n1-5-vhh gene in order to generate homo-
260
bivalent N1-3-VHHb and N1-5-VHHb expression constructs, which also contained a carboxy-
261
terminal hexahistidine tag (Fig. 1B). These bivalent constructs were expressed and purified from
262
the periplasmic fraction of E. coli as outlined above. We used an irrelevant bivalent in tandem
263
fused VHH specific for bacterial beta lactamase (BL-VHHb) as a negative control (52) (kindly
264
provided by Dr. Trong Nguyen-Duc, VUB, Brussels). Endotoxin levels of the bivalent VHH
265
preparations were determined as above.
266
Production of bivalent N1-VHHb fused to the mouse IgG2 Fc fragment (N1-VHH-Fc) in
267
seeds of stably transformed Arabidopsis thaliana lines
268
The three different n1-vhh-fc sequences coding for N1-3VHHm, N15-VHHm and N1-7-VHHm
269
fused at the aminoterminal region with the 2S2 signal peptide and at the carboxyterminal region
270
with the Fc region of mouse IgG2a and the KDEL retention signal were synthesized
271
commercially (GenScript), and cloned into a PphasGW binary T-DNA vector, resulting in the
272
PphasGWn1-vhh-fc vector (Fig. 1B). The Coronavirus GP4 subunit protein fused to the same
273
mouse IgG2a Fc moiety (GP4-Fc, kindly provided by Robin Piron, VIB and Ghent University)
274
was used as an irrelevant control in the antiviral assays. Agrobacterium C58C1RifR (pMP90)
275
was transformed with PphasGWn1-3-vhh-fc, PphasGWn1-5-vhh-fc, PphasGWn1-7-vhh-fc or
276
PphasGWGP4-fc constructs.
277
Agrobacterium
278
PphasGWn1-5-vhh-fc, PphasGWn1-7-vhh-fc and PphasGWGP4-fc binary T-DNA vectors and
279
the resulting strains were used for Arabidopsis thaliana floral dip transformation (53, 54). T1 to
280
T3 generations were produced and screened for best N1-VHH-Fc expressers. For protein
281
purification, 1 g of transformed A. thaliana seeds was crushed in 25 ml of 50 mM Tris-HCl, pH
282
8.0, 200 mM NaCl, 5 mM EDTA, 0.1% (v/v) Tween 20 and Complete protease inhibitor tablets
C58C1RifR (pMP90)
was
transformed
with
the
PphasGWn1-3-vhh-fc,
14
283
(Roche). The seed extracts were clarified by centrifugation and applied to a protein G
284
sepharose column (GE Healthcare). N1-VHH-Fc proteins were eluted in 0.1 M glycine pH 3.0
285
and immediately neutralized with 1 M Tris-HCl, pH 9.0. The recovery was between 1 and 10 mg
286
of N1- VHH-Fc. Endotoxin levels of the VHH-Fc preparations were determined as above.
287
Plaque assays
288
Monolayers of MDCK cells were grown in DMEM supplemented with 10% fetal calf serum, 1%
289
penicillin + streptomycin and 1% glutamine and non-essential amino acids at 37°C in 5% CO2.
290
MDCK TMPRSS2 cells (kindly provided by Dr. Wolfgang Garten, Philipps-Universität Marburg,
291
Germany) were cultured in DMEM supplemented with the above as well as geneticin (0.3
292
mg/ml) and puromycin (2 µg/ml). Expression of TMPRSS2 was induced with doxycycline (0.5
293
µg/ml) (55). At 70% confluence, the MDCK cells were infected at a multiplicity of infection (moi)
294
of 10 with NIBRG-14 ma, NIBRG-23 or H5N1 H274Y ma. Samples containing monovalent N1-
295
3/5/7-VHHm, bivalent N1-3/5-VHHb, bivalent N1-3/5/7/GP4-VHH-Fc, Oseltamivir or control
296
medium were mixed with 0.8% Avicel RC-591™ as overlay (56). After 2 to 4 days of incubation,
297
the cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were permeabilized
298
with 20 mM glycine containing 0.5% Triton X-100 (v/v). After blocking with BSA, the cells were
299
incubated for 2 h with polyclonal α-NIBRG-14 (1:1000) or anti-M2e monoclonal antibody
300
(1:5000). After washing, α-mouse whole IgG-HRP was used to visualize plaques with the
301
substrate TrueBlue™ Peroxidase Substrate (KPL).
302
Mouse experiments
303
All mouse experiments were conducted according to national (Belgian Law 14/08/1986 and
304
22/12/2003, Belgian Royal Decree 06/04/2010) and European legislation (EU Directives
305
2010/63/EU, 86/609/EEG) on animal regulations. All experiments on mice were approved by the
306
ethics committee of Ghent University (permit number LA1400091) and efforts were made to
15
307
avoid or diminish suffering of the animals. Specific-pathogen–free female BALB/c mice, 7–9
308
weeks old, were purchased from Charles River (Germany) and used in all experiments. Mice
309
were housed in ventilated cages with high-efficiency particulate air filters in temperature-
310
controlled, air-conditioned facilities and had food and water ad libitum. Mice were anaesthetized
311
by intraperitoneal injection of xylazine (10 μg/g) and ketamine (100 μg/g) before intranasal
312
administration of N1-VHHm, N1-VHHb or N1-VHH-Fc. The N1-VHHm in any format were diluted
313
in endotoxin-free phosphate-buffered saline (PBS) with 1% (w/v) bovine serum albumin, and
314
applied intranasally in doses ranging from 0.25 to 5 mg/kg, as indicated in the figure legends.
315
Mice were challenged 4 or 24 h later, as indicated, with 6 LD50 (H5N1 H274Y ma) or 4 LD50
316
(NIBRG-14 ma) of virus (50 μl, divided equally between the nostrils). A 30% loss in body weight
317
was the end point at which sick mice were euthanized. On day 4 after challenge, lung
318
homogenates were prepared in PBS, cleared by centrifugation at 4°C, and used for virus
319
titration. Monolayers of MDCK cells were infected with 50 μl of serial 1:10 dilutions of the lung
320
homogenates in a 96-well plate in serum-free Dulbecco's modified Eagle medium (Invitrogen)
321
supplemented with L-glutamine, non-essential amino acids, penicillin and streptomycin. After 1
322
h, the inoculum was replaced by medium containing 2 μg/ml of L-(tosylamido-2-phenyl) ethyl
323
chloromethyl ketone–treated trypsin (Sigma-Aldrich). End-point virus titers were determined by
324
hemagglutination of chicken red blood cells and expressed as TCID50 per milliliter. IAV RNA
325
levels were determined by quantitative RT-PCR. RNA was isolated from 150 μl of cleared lung
326
homogenate using the Nucleospin RNA virus kit (Machery-Nagel). The relative amount of
327
NIBRG-14 ma genomic RNA was determined by preparing viral cDNA and performing
328
quantitative PCR with M-genomic segment primers (CGAAAGGAACAGCAGAGT and
329
CCAGCTCTATGCTGACAAAATG) and probe (GGATGCTG) (probe no. 89; Universal
330
ProbeLibrary, Roche) and the LightCycler 480 Real-Time PCR System (Roche).
16
331
Selection and characterization of H5N1 N1-VHHm escape mutant viruses
332
NIBRG-14 ma virus was serially diluted and used to infect MDCK cells in the presence of N1-3-
333
VHHm (4.2 µM) or N1-H5-VHHm (3.7 µM), a dose corresponding to ten-fold the IC50 (Table 2).
334
Escape mutants were screened by monitoring cytopathic effects. After 9 passages in the
335
presence of N1-3-VHHm or N1-5-VHHm, escape viruses were plaque purified by growth on
336
MDCK cells overlaid with 0.6% low-melting agarose in the presence of N1-3-VHHm or N1-5-
337
VHHm. Escape virus isolates were amplified on MDCK cells, still in the presence of a ten-fold
338
IC50 of N1-3-VHHm or N1-5-VHHm. Total RNA was extracted from the supernatant and used to
339
clone the cDNA corresponding to the NA viral RNA segment (57). Deduced amino acid
340
substitutions in the NA sequence of escape viruses were modeled with PyMol (Delano
341
Scientific,
342
A/Vietnam/1194/2004 (PDB code 2HTY). N1-VHH binding assays were performed as described
343
(35). Briefly, 4-8 HA unit of wild type or plaque purified candidate escape viruses were used to
344
coat ELISA plates. After blocking, wells were incubated with different concentrations of N1-3-
345
VHHm, N1-3-VHHb, N1-5-VHHm or N1-5-VHHb. Binding of N1-VHHs was revealed by
346
incubation with an anti-His tag monoclonal antibody, followed by a secondary sheep anti-mouse
347
IgG antibody conjugated to HRP and visualized by addition of TMB substrate and absorbance
348
was measured at 450/655 nm.
349
Statistical analysis
350
Graphpad software (Graphpad Prism, version 6) was used for statistical analysis. For
351
comparing two groups in the fetuin and AVINA assays, t test was used. Differences between
352
mouse groups were tested using two-way ANOVA. When this test demonstrated a significant
353
difference between groups (P < 0.05), t tests were used to compare two groups on each day.
354
Kaplan-Meier survival curves were plotted and differences in survival were analyzed by the
355
Mantel-Cox log-rank test.
http://www.pymol.org)
using
the
H5N1
NA
structure
derived
from
17
356
RESULTS
357
Production of recombinant tetrameric neuraminidase
358
A Baculovirus Expression Vector System was used to produce recombinant soluble tetrameric
359
and enzymatically active H5N1 NA derived from A/crested eagle/Belgium/01/2004 (N1rec). The
360
N1rec coding sequence was cloned into a pAcMP2 expression vector (pAcMP2n1rec), under
361
the control of the AcNPV basic protein promoter that is active in the late phase of the
362
Baculovirus infection cycle (Fig. 1A). Infection of Spodoptera frugiperda (Sf9) cells with the
363
recombinant Baculovirus AcNPVN1rec resulted in sialidase activity in the cell supernatant,
364
suggesting that the N1rec product was soluble and enzymatically active NA (Fig. 1A). N1rec
365
protein was purified from the supernatant of infected Sf9 cells by three successive
366
chromatography steps, including size exclusion chromatography to collect tetrameric N1rec (see
367
Materials and Methods). Using MUNANA as a substrate, we calculated that purified N1rec had
368
a specific activity of 56,851 NA units/mg.
369
Immunization and VHH phage library construction
370
N1rec was used as an antigen for the generation and selection of NA-specific VHH. From an
371
immunized alpaca, we obtained a VHH phage library of 2 X 108 independent transformants,
372
57% of which had a cDNA insert of the correct size (data not shown). Seventy-eight positive
373
clones were selected, 13 of which had unique VHH sequences. The complementarity
374
determining region 3 (CDR3) had the most variable amino acid sequence among the 13 N1rec-
375
binding VHHs (N1-VHHm) (Fig. 1C). In silico structure prediction of the monomeric N1-VHHm
376
candidates showed that the CDR3 of N1-1-VHHm, N1-3-VHHm, N1-4-VHHm, N1-5-VHHm and
377
N1-6-VHHm had an electronegative protruding paratope (Fig. 1D).
378
Production and characterization of soluble monomeric N1-VHHm
18
379
The binding of the N1-VHHm proteins to N1rec was assessed by ELISA and compared with
380
binding to the recombinant M2e-tGCN4 protein, which like N1rec contains the same
381
tetramerizing leucine zipper (Table 1 and Fig. 1E). Except for N1-11-VHHm and N1-13-VHHm,
382
none of the N1-VHHm bound substantially to recombinant M2e-tGCN4 protein. We next used
383
the MUNANA substrate to assess the ability of the N1-VHHm candidates to inhibit the
384
enzymatic activity of N1rec. Four candidates, N1-1-VHHm, N1-3-VHHm, N1-5-VHHm and N1-6-
385
VHHm, inhibited the N1rec catalytic activity (Table 1). N1-3-VHHm and N1-5-VHHm inhibited
386
N1rec in the sub-micromolar range (Table 2), and together with N1-7-VHHm, they were
387
analyzed by SPR to determine their affinity for immobilized N1rec. Both N1-3-VHHm and N1-5-
388
VHHm had a high affinity for N1rec, with an equilibrium dissociation constant (KD) in the low
389
nanomolar (N1-5-VHHm) to high picomolar (N1-3-VHHm) range. N1-7-VHHm showed
390
approximately 10- to 100-fold lower affinity for N1rec than N1-5-VHHm and N1-3-VHHm,
391
respectively (Table 1). These binding affinities of N1-3-VHHm and N1-5-VHHm for N1rec
392
resemble the affinities reported for a monomeric VHH that inhibits the activity of lysozyme (33).
393
Competitive SPR analysis also showed that prior binding of N1-3-VHHm to N1rec abolished
394
subsequent binding of N1-1-VHHm, N1-5-VHHm and N1-6-VHHm. Conversely, N1-7-VHHm
395
binding to N1rec was not affected by prior incubation of N1rec with N1-3-VHHm or N1-5-VHHm
396
(Fig. 1F). This suggests that N1-3-VHHm and N1-5-VHHm bind an overlapping epitope within
397
N1rec, or that binding of N1-3-VHHm sterically hinders binding of N1-5-VHHm, while N1-7-
398
VHHm binds a different epitope.
399
We next analyzed the antiviral potential of N1-3-VHHm and N1-5-VHHm using the H5N1 strain
400
NIBRG-14 ma. The amino acid sequences of the NA of NIBRG-14 ma (derived from
401
A/Vietnam/1194/2004) and N1rec (derived from A/crested eagle/Belgium/01/2004) are highly
402
homologous, with only 13 amino acid differences, none of which map in or close to the NA
403
catalytic site. In the presence of N1-3-VHHm or N1-5-VHHm, the size of NIBRG-14 ma plaques
19
404
was reduced in a concentration dependent manner, and with IC50 values in low nanomolar
405
range (Table 2). In contrast, N1-7-VHHm did not affect the size or number of NIBRG-14 ma
406
plaques (Table 2). These results indicate that the in vitro antiviral potential of NA-specific VHH
407
proteins depends on their NA-inhibitory activity.
408
Bivalent N1-VHHb formats have enhanced NA inhibitory and antiviral potential
409
It has been reported that generation of multivalent formats of VHHs increases their functional
410
affinity by introducing avidity (34). For example, the introduction of avidity drastically decreased
411
the dissociation constant (Koff) of the VHH molecules directed against lysozyme, and
412
significantly improved their enzyme inhibitory activity over their monovalent format (51). To
413
increase the avidity of the N1-VHHm, we compared two different bivalent formats. In one
414
approach, we used the llama IgG2c hinge (17 amino acid residues) as a flexible linker to fuse
415
two identical N1-VHHm gene moieties in tandem, resulting in the bivalent n1-3-vhhb and n1-5-
416
vhhb expression elements (Fig. 1B and 2A). Despite repeated attempts, we could not generate
417
an N1-7-VHHb expression plasmid. The corresponding proteins were expressed and purified
418
from E. coli and their in vitro N1-VHHb NA-inhibitory and antiviral activities were compared with
419
those of their monovalent counterparts. N1rec inhibition by N1-3-VHHb and N1-5-VHHb was
420
2000- and 500-fold stronger than the corresponding N1-3-VHHm and N1-5-VHHm formats,
421
respectively (Table 2). Surprisingly, in a plaque assay using MDCK cells infected with NIBRG-
422
14 ma virus, both N1-3-VHHb and N1-5-VHHb seemingly had antiviral activities that were
423
comparable to the activities of their monovalent counterparts (data not shown). In this assay,
424
exogenous trypsin is used to facilitate maturation of HA in newly produced virions to allow
425
multicycle replication of the recombinant NIBRG-14 ma virus. We noticed that the llama IgG2c
426
hinge linker used to generate bivalent VHH formats was sensitive to trypsin cleavage (Fig. 2A).
427
To circumvent the use of exogenously added trypsin, we took advantage of TMPRSS2 MDCK
428
cells, which are transformed with the doxycycline-inducible serine protease TMPRSS2 and
20
429
allow multicycle replication of IAV in the absence of exogenous trypsin (55). Using monolayers
430
of TMPRSS2 MDCK cells for infection with NIBRG-14 ma, the antiviral effect of N1-3-VHHb and
431
N1-5-VHHb was increased 240- and 58- fold, respectively, compared with their monovalent
432
formats (Table 2). These results obtained with N1-VHHb indicate that there is a significantly
433
enhanced NA-inhibitory and antiviral activity of both bivalent N1-VHHb compared with the
434
monovalent N1-VHHm formats.
435
We next engineered N1-VHH variants fused to a mouse IgG2a-derived Fc domain as an
436
alternative bivalent format (N1-Vhh-Fc). To produce these more complex proteins, we used a
437
robust plant seed-based expression system with reported high yield of recombinant antibodies
438
(58, 59). For this, the n1-3-vhh, n1-5-vhh and the n1-7-vhh genes were fused to the sequence
439
encoding the hinge and Fc tail of mouse IgG2a (n1-vhh-fc). These n1-vhh-fc constructs were
440
cloned into the binary vector Pphas as a T-DNA expression cassette (Fig. 1B and 2B).
441
Subsequently, transgenic Arabidopsis thaliana plants were generated by Agrobacterium-
442
mediated floral dip transformation. Seed extracts of segregating T3 Arabidopsis transformants
443
were screened by ELISA using antibodies against the Fc part for the best expressers of the
444
recombinant dimeric N1-VHH-Fc proteins. N1-3-VHH-Fc and N1-5-VHH-Fc accumulated to high
445
levels in several transformants, and levels of 16 % of total soluble seed protein were found. On
446
the contrary, N1-7-VHH-Fc did not accumulate well and the best expressors contained about
447
1% VHH-Fc of total soluble seed protein (data not shown). A good correlation was observed
448
between the ELISA result and NA inhibitory activity in crude seed extracts from different T3
449
transformants (data not shown). N1-3-VHH-Fc, N1-5-VHH-Fc and N1-7-VHH-Fc were purified
450
from seed extracts by protein G affinity chromatography. Reducing SDS-PAGE and Coomassie
451
staining showed that the N1-VHH-Fc monomers migrated at approximately 42 kDa (Fig. 2B).
452
N1-3-VHH-Fc and N1-5-VHH-Fc had stronger N1rec inhibition capacity and in vitro antiviral
453
activity than their monovalent N1-3-VHHm and N1-5-VHHm counterparts (Table 2). Bivalent N1-
21
454
7-VHH-Fc bound to N1rec in ELISA assays (data not shown), but it failed to inhibit N1rec and
455
the H5N1 viruses in vitro. Taken together, these results indicate that the in vitro NA inhibition
456
and antiviral activity against NIBRG-14 ma virus is enhanced at least 30-fold by using bivalent
457
N1-VHHb and N1-VHH-Fc formats, compared with the monovalent N1-VHHm domain.
458
In vitro antiviral activity of N1-VHH against H5N1 clade 2.2, H5N1 Oseltamivir-resistant
459
and pandemic H1N1 2009 virus
460
Like NA from NIBRG-14, N1rec NA (derived from A/crested eagle/Belgium/01/2004) belongs to
461
the clade 1 of the H5N1 NAs. To assess the potential cross reactivity of the NA activity inhibition
462
(NAI) of the isolated VHHs in the three available formats (N1-VHHm, N1-VHHb and N1-VHH-
463
Fc), we focused first on NIBRG-23 NA (derived from A/turkey/Turkey/01/2005), a clade 2.2
464
H5N1 virus (60). Although there are only a few laboratory-confirmed human infections with
465
H5N1, it appears that clade 2 H5N1 viruses are responsible for most of these zoonotic
466
infections with HPAIV (61). N1-3-VHHm and N1-5-VHHm reduced in vitro growth of NIBRG-23
467
virus with an IC50 in the low micromolar range (Table 2). N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc
468
and N1-5-VHH-Fc displayed about 150-fold higher in vitro antiviral activity compared to the
469
corresponding monovalent N1-VHHm against this clade 2 virus, as judged by a plaque size
470
reduction assay. Based on these findings, we conclude that the two NA-inhibitory VHHs inhibit
471
in vitro replication of representative H5N1 IAV from clades 1 and 2 with a comparable efficiency,
472
suggesting that they target a common epitope shared by the NA of these viruses.
473
Oseltamivir-resistant IAV frequently emerges and spreads in the human population. Several
474
mutations have been reported to contribute to Oseltamivir-resistance, but among them the
475
H274Y mutation (N2 numbering) is the most common found in Oseltamivir-resistant viruses
476
(62). We used reverse genetics to generate a clade 1 H5N1 virus harboring the H274Y mutation
477
in NA (derived from A/crested eagle/Belgium/01/2004), while the HA segment was derived from
22
478
NIBRG-14 and the remaining six segments from PR/8, resulting in H5N1 H274Y virus. Next, we
479
determined if N1-3-VHHm, N1-5-VHHm, N1-3-VHHb, N1-5-VHHHb, N1-3-VHH-Fc and N1-5-
480
VHH-Fc can inhibit this virus. As so far all N1-3-VHH formats performed alike N1-5-VHH formats
481
in vitro, we were surprised that only N1-3-VHH (monovalent and both bivalent formats), but not
482
N1-5-VHH in any format, reduced the growth of H5N1 H274Y (Table 2). Compared to NIBRG-
483
14 ma, the H5N1 H274Y IC50 values were three- to seven-fold higher, but still in the low
484
nanomolar range. Even though the competitive SPR analysis suggested that the N1-3-VHHm
485
and N1-5-VHHm epitopes in NA overlap (Fig. 1F), the contact residues necessary for their
486
binding are probably not identical. We conclude that the three evaluated formats of N1-3-VHH
487
can inhibit growth of H5N1 H274Y viruses in vitro, and that both bivalent formats have
488
enhanced in vitro antiviral activity. In addition, the H274Y mutation seems to be sufficient to
489
abolish the in vitro antiviral effect of the N1-5-VHH formats.
490
Next, we tested the antiviral potential of all N1-3-VHH and N1-5-VHH formats against a
491
pandemic H1N1 2009 virus isolate (pH1N1). We used fetuin and MUNANA (AVINA assay) as
492
two alternative substrates for virion-associated NA activity. Using NIBRG-14 ma, and based on
493
MUNANA hydrolysis, monovalent N1-3-VHHm and N1-5-VHHm had significant inhibitory
494
activity, compared with the negative controls SA-VHHm (P < 0.05) and PBS (P < 0.01), although
495
this tendency was not significant in the fetuin assay (Fig. 3A). We attribute these differences
496
between the two assays to the type of substrate used: small molecule MUNANA versus fetuin,
497
which is a large protein carrying multiple sialylated glycans. In the case of fetuin, HA plays an
498
important role in recruiting NA to the substrate, which may alter the accessibility of the N1-VHHs
499
to the NA target. We found that the bivalent molecules N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc
500
and N1-5-VHH-Fc significantly inhibited NA activity for both substrates (P < 0.01, Fig. 3A).
501
In line with our previous results using H5N1 H274Y IAV, only N1-3-VHHb and N1-3-VHH-Fc
502
showed NAI activity using both substrates. (P < 0.05, or P < 0.01) (Fig. 3B). N1-5-VHHb and
23
503
N1-5-VHH-Fc showed inhibitory potential against pH1N1 in both assays. (P < 0.05) (Fig. 3C).
504
We could not detect binding of the N1-VHHs to A/NewCaledonia/20/99 or inhibition of NA
505
activity of this virus (Fig. 3D and data not shown). These AVINA and fetuin results for NIBRG-14
506
ma and H5N1 H274Y NA are in accordance with the N1rec inhibition and the plaque size
507
reduction assays mentioned before (Table 2). In addition, both bivalent N1-5-VHHb formats
508
inhibited pH1N1 virion-associated NA activity, suggesting a degree of intra subtype inhibitory
509
effect of the N1-VHHs.
510
N1-3-VHHm and N1-5-VHHm treatment reduce early morbidity, but not viral lung titer, in
511
H5N1-challenged mice
512
We next evaluated the in vivo antiviral effect of N1-3-VHHm and N1-5-VHHm. The endotoxin
513
levels of the VHH preparations that were used in vivo were low and comparable for the NA-
514
specific VHH proteins and their irrelevant control counterparts (Table 3). In the first experiment
515
we administered intranasally 100 µg of N1-3-VHHm, N1-5-VHHm or N1-7-VHHm to BALB/c
516
mice 4 hours before challenge with 4 LD50 of NIBRG-14 ma. Two groups were included as
517
positive controls (i) a group of mice that received 30 µg of H5-VHHb, a bivalent NIBRG-14 ma
518
HA neutralizing VHH (36), and (ii) a group of mice that received daily oral administration of
519
Oseltamivir at a high dose (45 mg/kg/day). Mice treated with N1-3-VHHm or N1-5-VHHm
520
showed significantly reduced morbidity at 72 and 96 h after infection, compared with the groups
521
treated with N1-7-VHHm and PBS (P < 0.001) (Fig. 4A). Four days after infection, the mice were
522
sacrificed to determine lung virus titers by endpoint dilution in a TCID50 assay. All treated groups
523
of mice (N1-3-VHHm, N1-5-VHHm, N1-7-VHHm, SA-VHHm, PBS and Oseltamivir), except the
524
H5-VHHb group, had a high lung virus load ranging from 104.75 to 106.48 TCID50/ml (data not
525
shown). We also measured the amount of viral RNA in the lung homogenates using a genome
526
strand-specific RT-qPCR method. Except for the samples derived from the H5-VHHb treated
527
mice (P < 0.001), all the N1-VHH treated groups showed high viral RNA levels 96 h after
24
528
infection, comparable to those in the PBS-treated group (Fig. 4B). NA activity in the lung
529
homogenates of the treated groups, measured by AVINA, indicated that there was no difference
530
between the N1-VHH and PBS treated groups, whereas NA activity was strongly reduced in the
531
H5-VHHb and Oseltamivir groups (Fig. 4C). These results are in line with the RT-qPCR results
532
and confirmed the presence of a high viral titer in the lung homogenates of the N1-VHH groups,
533
despite low morbidity. We speculate that the N1-VHH treatment did not have a major impact on
534
virus entry in this in vivo challenge model, but rather reduced virus spread within the lung
535
compartment. A recent systems analysis of influenza A virus-associated pathology in the
536
mouse, revealed a positive correlation between virus spread in the lung tissue and high
537
pathogenicity irrespective of the number of infectious virus particles that were produced (63). In
538
addition, shearing forces that are generated during the preparation of lung homogenates may
539
have released virions from the cell membranes, of which the release was hindered in the intact
540
lung by NA-inhibitory nanobodies. We conclude that intranasal administration of N1-3-VHHm
541
and N1-5-VHHm prevents body weight loss after challenge with NIBRG-14 ma virus during the
542
early stage of H5N1 IAV infection, without apparent decrease in viral titer in the lung
543
homogenates.
544
Protection by bivalent N1-VHHb against H5N1 challenge
545
The in vitro results indicated that the potency of the bivalent formats of the inhibitory N1-VHHs
546
against the tested H5N1 viruses was 30- to 240-fold higher than their monovalent counterparts
547
(Table 2). We therefore assessed if this heightened antiviral effect would also be reflected in an
548
in vivo challenge experiment. We first determined the protective potential during the early
549
stages of H5N1 infection. Four hours before challenge with 4 LD50 of NIBRG-14 ma, groups of
550
BALB/c mice were intranasally given 60 µg of N1-3-VHHb, N1-5-VHHb, or BL-VHHb (a bivalent
551
VHH directed against the irrelevant bacterial target β-lactamase), and 84 µg of N1-3-VHH-Fc,
552
N1-5-VHH-Fc, or GP4-Fc (a plant-produced Coronavirus GP4-Fc fusion protein). Treatment with
25
553
N1-3-VHHb or N1-3-VHH-Fc significantly improved morbidity 72 and 96 h after infection (hpi)
554
compared with the negative control groups GP4-Fc and PBS (P < 0.001) (Fig. 4D). The
555
protection against weight loss was comparable to that observed with the positive controls (H5-
556
VHHb and Oseltamivir, the latter given daily at 45 mg/kg/day by gavage). Treatment with the
557
bivalent formats of N1-5-VHH resulted in reduction of morbidity that was significant at 60, 72
558
and 96 hpi compared with the negative controls (P < 0.001) (Fig. 4D). Determination of the lung
559
virus load on day four after challenge revealed that all challenged groups, except the H5-VHHb-
560
treated mice (no virus detectable), had high and comparable lung virus loads (data not shown).
561
In addition, the NA activity in the lung homogenates of the groups treated with N1-VHH bivalent
562
formats was assessed by an AVINA assay. The lung homogenates treated with bivalent N1-
563
VHHs (N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc and N1-5-VHH-Fc) showed 2-5 fold decrease in
564
NA activity when compared to the BL-VHHb (P < 0.001), GP4-Fc (P < 0.05, or P < 0.01) and
565
PBS treated groups (P < 0.01) (Fig. 4E). This could be attributable to residual inhibitory bivalent
566
N1-VHHb in the lung preparation, possibly due to an increased half life of the bivalent N1-VHHs,
567
relative to N1-VHHm, for which no significant reduction in lung associated NA activity was
568
demonstrated (Fig. 4C). Taken together, treatments with bivalent formats of N1-3-VHH and N1-
569
5-VHH improve protection during the first four days following NIBRG-14 ma challenge and
570
reduce the lung associated NA activity, a surrogate for viral load, in H5N1-infected mouse lungs.
571
Dose-dependent protection against H5N1 challenge
572
To test if prophylactic administration of N1-3-VHHb, N1-3-VHH-Fc, N1-5-VHHb or N1-5-VHH-Fc
573
can protect mice against a lethal (4 LD50) challenge with NIBRG-14 ma, we repeated the
574
protection studies and followed the mice for two weeks. We focused on the most potent N1-
575
VHHb bivalent formats and assessed their protective efficacy in a dose-response experiment.
576
Groups of four BALB/c mice were treated intranasally with 60, 12, 2.5 or 0.5 µg of N1-3-VHHb
577
or N1-5-VHHb. In parallel, another group was treated daily by oral administration of Oseltamivir
26
578
(45 mg/kg/day). Negative control groups were treated with 60 µg of BL-VHHb or with PBS
579
before challenge. Mice that had received 60 µg of N1-3-VHHb, N1-5-VHHb or BL-VHHb before
580
the challenge also received a second intranasal dose of 60 µg of the same bivalent VHH on day
581
6 after the challenge. All mice from the PBS and BL-VHHb treatment groups died 9-10 days
582
after infection (Fig. 5C and D). In contrast, treatment with Oseltamivir and intranasal high dose
583
N1-3-VHHb or N1-5-VHHb (60 µg) protected the mice against lethality. In agreement with this,
584
Oseltamivir and N1-5-VHHb (60 µg) reduced post-challenge body weight loss compared to
585
control treated mice (Fig. 5A and B). Although administration of the highest amount of N1-3-
586
VHHb prevented death, it did not affect body weight loss. A single intranasal dose of 12 µg or
587
2.5 µg of N1-3-VHHb or N1-5-VHHb provided partial protection against mortality but failed to
588
reduce morbidity (Fig. 5A-D).
589
We next evaluated protection against a potentially lethal challenge with NIBRG-14 ma by prior
590
single dose intranasal administration of the plant-produced N1-3-VHH-Fc and N1-5-VHH-Fc
591
formats. N1-3-VHH-Fc (80 µg and 17 µg) as well as Oseltamivir prevented death following
592
challenge with NIBRG-14 ma (Fig. 6C), whereas treatment with 3.5 µg of N1-3-VHH-Fc
593
prevented death of some animals. However, challenge was associated with significant body
594
weight loss for all doses and treatments (Fig. 6A and 6B).
595
When comparing the protective efficacy of the two bivalent formats of N1-3-VHH and N1-5-
596
VHH, it appeared that the Fc moiety in the N1-VHH-Fc formats provides an extra protective
597
effect against morbidity (compare 60 µg N1-3-VHHb with 84 µg N1-3-VHH-Fc in figures 5A and
598
6B) as well as against mortality following NIBRG-14 ma challenge (Fig. 5 and 6). We conclude
599
that a single dose of both bivalent N1-3/5-VHH formats can protect mice against a potentially
600
lethal challenge with an H5N1 virus, and that Fc-fused VHHs provide better protection,
601
suggesting that Fc Receptor binding molecules contribute to protection.
27
602
N1-3-VHHb and N1-3-VHH-Fc protect against challenge with Oseltamivir-resistant H5N1
603
Our in vitro analysis demonstrated that monovalent and bivalent formats of N1-3-VHH, but not
604
N1-5-VHH, reduced the growth of Oseltamivir-resistant H5N1 H274Y virus (Table 2). To
605
evaluate these findings in vivo, groups of six BALB/c mice received 30 µg of N1-3-VHHb, N1-5-
606
VHHb or BL-VHHb by intranasal administration 24 h before challenge with 4 LD50 of NIBRG-14
607
ma or 6 LD50 of H5N1 H274Y ma. In parallel, a group was treated by daily oral administration of
608
Oseltamivir (1 mg/kg/day), a dose that has been reported to fully protect laboratory mice against
609
challenge with highly pathogenic H5N1 (64). In line with our previous results, mice treated with
610
N1-3-VHHb, N1-5-VHHb or Oseltamivir suffered from substantial but transient weight loss, but
611
they survived challenge with NIBRG-14 ma virus, whereas mice receiving PBS or BL-VHHb
612
died before day 10 after challenge (Fig. 7). This result demonstrates that a single intranasal
613
administration of N1-3-VHHb or N1-5-VHHb is sufficient to protect against a subsequent lethal
614
challenge (24 h later) with an H5N1 virus. Following challenge with H5N1 H274Y virus, the N1-
615
3-VHHb treated mice were severely sick, but all mice survived (Fig. 7A and 7C). All other
616
groups, including the Oseltamivir treated mice, died after challenge with the H5N1 H274Y virus
617
by day 10 after infection (Fig. 7A and 7C). Taken together, bivalent N1-3-VHHb treatment of
618
mice before challenge with an Oseltamivir-resistant H5N1 virus does not control morbidity
619
adequately but results in full recovery and survival. Furthermore, bivalent N1-5-VHHb does not
620
protect mice against this challenge, suggesting that the N1-VHHb formats (i.e. tandem repeats
621
of VHH separated by a short linker) require the NA inhibitory activity for in vivo protection.
622
Finally, we compared the protective potential of intranasally instilled N1-3-VHH-Fc, N1-5-VHH-
623
Fc and N1-7-VHH-Fc in our lethal challenge model. Following challenge with 4 LD50 of NIBRG-
624
14 ma, all mice in the N1-3-VHH-Fc and Oseltamivir groups lost body weight up until day 7 after
625
challenge (Fig. 7B) and then started to recover, and all mice survived this challenge (Fig. 7D). In
626
contrast, all PBS or GP4-Fc treated mice, except one, became morbid and died by 10 days post
28
627
infection (Fig. 7B and 7D). In the N1-7-VHH-Fc group, only one out of six mice died after
628
challenge. This is very surprising because N1-7-VHHm and N1-7-VHH-Fc have no detectable
629
antiviral activity in vitro. Remarkably, in this experiment mice that had received N1-5-VHH-Fc
630
did not lose weight after challenge with NIBRG-14 ma (Fig. 7B). A challenge with 6 LD50 of
631
H5N1 H274Y virus was lethal to all mice except those that had been pretreated with N1-3-VHH-
632
Fc (all mice survived) or N1-7-VHH-Fc (four out of six mice survived), though all animals lost
633
substantial body weight (Fig. 7B and 7D). We conclude that protection against a potentially
634
lethal challenge with Oseltamivir-resistant H5N1 virus can be obtained by a single dose of N1-3-
635
VHH-Fc or with N1-7-VHH-Fc, although the latter construct is less potent. This indicates that the
636
IgG2a Fc moiety contributes to protection, even in the absence of NA inhibitory activity.
637
N1-5-VHHm escape mutants
638
To identify the amino acid residues involved in the binding of N1-3-VHHm and N1-5-VHHm to
639
H5N1 NA, escape viruses were selected and plaque purified after serial passage of NIBRG-14
640
ma virus in MDCK cells, in the presence of 10 IC50 of N1-3-VHHm or N1-5-VHHm, essentially as
641
described before (35). After nine passages, candidate escape mutants were plaque purified and
642
the NA sequences of four (N1-5-VHHm selection) and three (N1-3-VHHm selection) isolated
643
escape viruses were determined. One of the escape viruses that was selected with N1-5-VHHm
644
contained a mutation in the NA coding region that resulted in a change from isoleucine to
645
threonine at position 437 in the deduced amino acid sequence. This substitution is located on
646
the NA surface, relatively close to the active site (Fig. 8A). A conserved NA epitope that is
647
recognized by HCA-2 under denaturing conditions (65, 66) and localized within the NA catalytic
648
site, and the amino acids necessary for binding a recently reported set of mAbs that recognize
649
H1N1 and H5N1 NA (67) do not overlap with this N1-5-VHHm escape mutations (Fig. 8A).
650
Sequence alignment of a set of representative N1 influenza A viruses, including human H1N1,
651
other H5N1 viruses and an avian H6N1 virus showed that I437 is conserved in these NA
29
652
sequences (Fig. 8B). Binding studies using immobilized virus revealed that the I437T escape
653
virus was no longer bound by N1-3-VHHm, N1-3-VHHb, N1-5-VHHm and N1-5-VHHb (Fig 8C).
654
This result is in accordance with the Biacore data, which showed that N1-3-VHHm and N1-5-
655
VHHm compete for binding to recombinant H5N1 NA (Fig. 1F) and suggests that I437 is part of
656
the VHH epitope in NA or stabilizes the conformation of that epitope.
657
Interestingly, the other three plaque purified N1-5-VHHm escape viruses and all three plaque
658
purified viruses that grew in the presence of N1-3-VHHm contained wild type NA. Possibly,
659
these viruses carry compensatory mutations in other viral proteins such as HA as was described
660
before for influenza A virus passaged in vitro in the presence of small molecule inhibitors of NA
661
(68). Taken together, H5N1 viruses that overcome the inhibition by N1-3-VHHm and N1-5-
662
VHHm can be selected in vitro and one way of escape is through the acquisition of a mutation in
663
NA that results in loss of binding to the VHHs.
664
30
665
DISCUSSION
666
NA Abs usually do not virus neutralizing and are thought to exert their antiviral effects further
667
downstream in the IAV infection process. Still, NA-specific antibodies can significantly reduce
668
viral replication and disease severity (69, 70). Ab titers against N1 or N2 have been associated
669
with high NA enzymatic activity in the vaccine preparation, suggesting that the NA content and
670
activity in IAV vaccines predicts immunogenicity (71). NA of human influenza viruses is subject
671
to antigenic drift at a pace that is comparable to that of HA (72). This suggests that NA is
672
subject to intense immune selection pressure, which is surprising given that antibodies directed
673
against NA do not neutralize the virus in vitro and that during natural infections immune
674
responses against HA are more pronounced than those against NA, presumably because HA
675
outnumbers NA on influenza virions. Biologically, HA and NA cooperate and antigenic variation
676
in NA is in part driven by antigenic escape selection pressure on HA (73). Nevertheless,
677
antibodies directed against NA remain underexplored as potential treatment against influenza.
678
Our N1-VHHs are not the first inhibitory mAbs described against H5N1 NA. Recently, the
679
IgG2bκ mAb 2B9 directed against clade 1 A/Vietnam/1203/04 NA showed interclade and
680
Oseltamivir-resistant mutant NAI in vitro (74). In NAI assays using MUNANA and NA-Star
681
substrates, the 2B9 mAb IC50 ranged from 1 to 8 µg/ml against H5N1 clade 1 and clade 2.1
682
virions, and against an Oseltamivir-resistant double mutant H274Y and N294S. In another
683
recent report on IgG2b mAbs targeting multiple N1 NA, intra-subtype mAbs showed a relatively
684
high H1N1 NAI potency (IC50 of 23.6 to 68.1 ng/ml), which was even lower when
685
A/Vietnam/1203/04 H5N1 NAI potency was assessed (1.41-2.42 µg/ml) (67). We used two
686
inhibitory N1-VHH formats to assess NA inhibitory and antiviral potential. Results from our
687
N1rec inhibition assays showed that N1-3-VHHm and N1-5-VHHm IC50 was 425 nM (6.3 µg/ml)
688
and 374 nM (5.6 µg/ml), respectively, and the introduction of bivalency (N1-VHHb and N1-VHH-
689
Fc) resulted in 2-3 log-fold further increase in their inhibitory potential (5.2-125 ng/ml) (Table 2).
31
690
The above mentioned conventional mAbs, which are bivalent, have an H5N1 NAI activity that is
691
similar to our N1-VHHm. We hypothesize that the much smaller size of VHH molecules
692
compared with conventional antibodies compensates for the lack of avidity that is present in
693
conventional antibodies to explain this.
694
Harmsen et al. reported the first VHH targeting neuraminidase (39). In contrast with our work,
695
where we used recombinant NA protein to immunize an alpaca, these authors obtained VHH by
696
immunizing llamas using whole IAV. This strategy yielded mainly HA and NP binders, and
697
required a cross-selection strategy with homologous NA, followed by further selection with
698
recombinant NA to obtain NA binders. This approach resulted in VHH binding to homologous
699
and non-homologous IAV, but only some of the NA cross-binding VHHs showed NA inhibition
700
activity (fetuin substrate). It is also important to point out that the recombinant NA activities used
701
by Harmsen et al. were 316 to 13.5 times lower than our N1rec specific activity (57
702
µmol/mg/min), while their VHH NA inhibition IC50 values ranged from 124 ng/ml to 2 ng/ml, using
703
homologous NA. The apparently lower IC50 values of the Harmsen VHHs (3 to 200 fold lower
704
than for our N1-VHHm) could therefore be explained by a 1-2 log lower NA specific subtype
705
activity. All together, these results suggest that a similar NAI potency range was obtained with
706
our monovalent N1-VHHm. In vitro, monovalent N1-3-VHHm and N1-5-VHHm had an IC50 that
707
was almost identical to that of Oseltamivir for N1rec and for the two Oseltamivir-sensitive H5N1
708
viruses we assayed (table 2).
709
In the MDCK TMPRSS2-based assay, which allowed us to assess multiple rounds of viral
710
replication in the absence of exogenous trypsin, the antiviral effect of bivalent VHH formats (N1-
711
VHHb and N1-VHH-Fc) was clearly enhanced compared with monovalent N1-VHHm molecules
712
(Table 2). For NIBRG-14 ma, the N1-VHH concentration necessary to reduce the plaque size by
713
50% (plaque assay IC50) for N1-3-VHHb and N1-5-VHHb was 7.6 nM (243 ng/ml) or 14.5 nM
714
(464 ng/ml) respectively, which are 240- and 58-fold lower than their monovalent formats. In
32
715
fact, we noticed a consistent 30-fold or higher inhibitory activity of bivalent N1-3-VHHb and N1-
716
3-VHH-Fc compared to monovalent VHHs in a biochemical assay using N1rec and against the
717
three H5N1 viruses tested in vitro. H5N1 H274Y was an exception in that N1-5-VHH mono or
718
bivalent formats failed to inhibit this Oseltamivir resistant virus (Table 2). These results also
719
suggest that the epitope recognized by N1-3-VHHm and N1-5-VHHm is conserved between the
720
NAs from clade 1 and 2 H5N1 viruses.
721
Intranasal administration of a relatively high dose of N1-3-VHHb or N1-5-VHHb (60 μg; 3.3
722
mg/kg) 24 h before H5N1 challenge protected mice against death, but significant body weight
723
was still lost (Fig. 5). In this challenge model, the N1-VHH-Fc formats protected better than the
724
N1-VHHb formats. A single dose of 17 μg (1.1 mg/kg, N1-3-VHH-Fc) or 3.5 μg (0.23 mg/kg, N1-
725
5-VHH-Fc) resulted in 100% survival of mice, and reduced morbidity compared to control
726
treated animals (Fig. 6 and 7). When comparing the molar ratios of N1-VHH-Fc (N1-3-VHH-Fc
727
and N1-5-VHH-Fc) with N1-VHHb (N1-3-VHHb or N1-5-VHHb), we found that approximately 10
728
to 50 times lower amounts of the N1-VHH-Fc formats were necessary to give 100% survival to
729
H5N1 challenged mice. For comparison, also in a mouse model, conventional mAb 2B9 resulted
730
in only partial (50%) protection against H5N1 A/Vietnam/1194/2004 challenge (74). Even so,
731
this required intravenous administration of 500 μg (33.3 mg/kg) of 2B9 1 h before 10 LD50 of
732
HPAIV H5N1, with daily boosts for 5 days. Furthermore, when we compared the 2B9 treatment
733
with our single-dose treatment with N1-VHHb or N1-VHH-Fc, 10- or 150-fold lower amount of
734
mAb, respectively, was required to rescue 100% of H5N1 challenged mice without any boost.
735
When comparing with a study by Wan et al., the lowest prophylactic single-dose of a
736
conventional IgG2a mAb necessary to fully rescue mice from a 10 LD50 H5N1 challenge was 15
737
mg/kg, whereas 0.23 mg/kg of our N1-5-VHH-Fc protected all mice from death after a 4 LD50
738
challenge
739
A/Vietnam/1194/2004) is almost identical to that used in the Wan report (A/Vietnam/1203/2004).
with
NIBRG-14
ma
(67).
The
target
NA
of
NIBRG-14
(derived
from
33
740
In both reports on NA mAbs discussed above (67, 74), the route of administration (intravenous
741
or intraperitoneal) could have been an important factor and it might help explain the apparent
742
superior protection of our N1-VHH formats, which we administered intranasally, since VHHs
743
withstand nebulization. Moreover, it has been shown that anti-RSV Nanobodies give extended
744
protection against viral infection in vivo when administered via a nebulizer. It may be expected
745
that optimization of the delivery will further help to improve N1-VHH efficacy. N1-VHHm
746
treatments did not significantly reduce the virus titers or viral genomic loads in lung
747
homogenates 4 days after infection (Fig. 4C). The weak, direct antiviral activity of our NA-
748
specific VHHs in the in vivo model is in line with the paradigm that NA Abs or NA-inhibitory
749
drugs do not prevent infection.
750
The observation that N1-5-VHHm, N1-5-VHHb and N1-5-VHH-Fc did not have any antiviral
751
activity against the Oseltamivir-resistant H5N1 H274Y virus in vitro was surprising since in the
752
SPR assay N1-3-VHHm and N1-5-VHHm bound to overlapping epitopes in NA or sterically
753
hindered each other’s binding (Fig. 1F). This discrepancy was also observed in vitro for N1-7-
754
VHHm, whereas N1-7-VHH-Fc did protect mice against challenge with H5N1 H274Y virus (Fig.
755
7). The latter observation indicates that protection by NA-based antibodies does not require the
756
NAI activity of these antibodies, provided that an Fc domain is present to contribute Fc
757
Receptor-dependent effector functions. The finding that also N1-7-VHH-Fc had in vivo antiviral
758
activity despite lack of NAI activity is in line with this (Fig. 7B and 7D). Mouse IgG2a is one of
759
the most potent Ab isotypes to induce antibody-dependent cellular cytotoxicity or complement-
760
dependent cellular cytotoxicity, due to its high affinity for FcγRIII and –IV and especially for
761
FcγRI, which binds exclusively to IgG2a. IgG2a has an intermediate affinity for FcγRIV that is
762
one order of magnitude higher than its affinity for the inhibitor receptor FcγRIIB (75).
763
The VHH CDR3 is a major region responsible for the contact of VHH with antigen (25, 76). All
764
NA inhibitory N1-VHHs reported here (N1-1-VHHm, N1-3-VHHm, N1-5-VHHm and N1-6-VHHm)
34
765
have a predicted electronegatively charged protruding paratope that corresponds to their CDR3
766
(Fig. 1C and 1D). The CDR3 sequences of N1-3-VHHm (HGXXHGHGXHX, 10 residues) and
767
N1-5-VHHm (HXHXHHXHXH, 12 residues), where "H" is a hydrophobic residue and "X" is an
768
aspartic or glutamic acid, might be involved in the direct contact with their epitope or epitopes.
769
When comparing N1-3-VHHm and N1-5-VHHm CDR3 sequences with the most potent NA
770
inhibitory VHHs reported by Harmsen et al., we found similarities in their CDR3 sequences. For
771
example, the CDR3 sequences of one N8-binding VHH (UHXHHHGHGUHHXUPHXH, 18
772
residues) and one N2-binding VHH (HHXHGHHGGHGHUHXH, 16 residues), where “U” is a
773
polar uncharged residue, shared similarities with the N1-3-VHHm and N1-5-VHHm CDR3s.
774
The available crystal structures of mAbs in complex with NA are all for the group 2 NA,
775
specifically N2 and N9 (77-80). These structures showed that these mAb epitopes rarely include
776
conserved amino acid residues from the catalytic site; rather, they target amino acids in loops
777
surrounding the NA catalytic site, also described as the major antigenic regions of NA. It seems
778
that the N1-5-VHHm epitope is partially shared with other N1 NA molecules, as the pandemic
779
H1N1 NA activity inhibition suggests (Fig. 3).
780
Plant-produced Abs have been considered a promising and effective alternative for Ab
781
production by recombinant yeast or mammalian cell culture systems. Our N1-VHH-Fc is the first
782
reported plant-produced Ab against an influenza antigen. Their scalability, robustness and
783
speed of production are major advantages, even though the glycosylation patterns of the plant-
784
produced Abs (rich in high mannose residues) are different from those of the Abs produced in
785
mammalian expression platforms (81). The use of plants for Ab production also presents an
786
interesting approach for developing countries: long storage life, relative ease of purification or
787
delivery, and use of established agricultural techniques are very attractive. To our knowledge,
788
only three single domain Abs produced in plants have been reported as stably transformed lines
789
(82-84), but their yields were very low. Results from our lab demonstrated that the Fc moiety
35
790
significantly improved VHH accumulation in seeds (compared to the monomeric VHH alone)
791
with expression levels of 10-20% of total seed proteins (54, 85).
792
HPAIV H5N1 remains a public health threat. The experimental adaptation of H5N1 virus for
793
airborne transmission in ferrets, with mutations in HA and PB2, warns of the possibility of
794
human-to-human transmission of zoonotic HPAIV (1, 2). Should the HPAIV H5N1 become
795
transmissible among humans, NA targeting could be part of an effective strategy to mitigate
796
disease caused by such a virus. In addition, an interesting prophylactic and therapeutic
797
approach that deserves further validation is the combination of VHH targeting IAV NA, HA or M2
798
proteins in cocktails or biparatopic bivalent VHH constructs that could diminish the antigenic drift
799
in IAV, and lead to much higher selective pressure against IAV VHH.
36
800
Acknowledgements
801
The authors gratefully appreciate the excellent technical support of Tine Ysenbaert, Frederick
802
Vervallle and Jonah Nolf. We thank Dr. Thierry Van den Bergh from the Veterinary and
803
Agrochemical Research Centre in Brussels (Belgium) for providing cDNA of Neuraminidase of
804
A/crested eagle/Belgium/01/2004 (H5N1) influenza virus, Dr. Wolgang Garten from Philipps-
805
Universität Marburg, Germany for MDCK TMPRSS2 cells, Dr. John Wood from the UK National
806
Institute for Biological Standards and Control for providing NIBRG-14 and -23 virus, Dr. Alan
807
Hay (MRC National Institute for Medical Research, Mill Hill, London, UK) for providing A/New
808
Caledonia/20/99 virus, and St. Jude Children's Research Hospital for providing the A/PR/8/34
809
based eight-plasmid system for generating recombinant influenza A viruses. FMC was funded
810
by a pre-doctoral grant from the Biotechnology Institute of Flanders (VIB) and supported by
811
IUAP BELVIR project p7/45.
812
37
813
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1120
44
1121
Figure 1. Characterization of monovalent N1-VHH candidates that bind recombinant H5N1-
1122
derived neuraminidase. A. Soluble N1rec derived from H5N1 A/crested eagle/Belgium/01/2004
1123
expressed in Sf9 cells. A diagram of the n1rec Baculovirus expression cassette (top panel);
1124
promoter, Baculovirus basic protein promoter; ssHA, secretion signal of HA; tGCN4,
1125
tetramerizing GCN4-derived leucine zipper; H5N1 NA, extracellular domain of NA (amino acid
1126
residues 53–449). The lower panel shows NA activity in the culture supernatants of different
1127
amounts of AcNPVN1rec-infected cells or mock-infected cells, using MUNANA as a substrate.
1128
RFU: relative fluorescent units. B. Schematic representation of the expression cassettes of
1129
N1rec-binding VHH domains and derived bivalent formats. Bacteria-produced monovalent N1-
1130
VHHm (n1-vhhm), with the amino-terminal periplasmic signal sequence (pelB), the n1-vhhm
1131
coding information, and a carboxy-terminal hexahistidine tag (his)(top panel). Bacteria-produced
1132
bivalent N1-VHHb with two n1-vhhm moieties fused by a flexible linker (llama IgG2c hinge) and
1133
a hexahistidine tag (his)(middle panel). For expression in transgenic Arabidopsis thaliana seeds,
1134
the N1-VHH-Fc insert was cloned into the seed-specific expression cassette of the PphasGW
1135
vector. LB (left border of T-DNA); 3’ ocs (3’ end of the octopine synthase gene); npt II (neomycin
1136
phosphotransferase II open reading frame); Pnos (nopaline synthase gene promoter); Pphas (β-
1137
phaseolin gene promoter); 5’ utr (5’ UTR of arc5-I gene); SS (signal sequence of the
1138
Arabidopsis thaliana 2S2 seed storage protein gene); n1-vhh-fc (coding sequence of the N1-
1139
VHH gene fused to mouse CH2 and CH3 IgG2a); KDEL (endoplasmic reticulum retention
1140
signal); 3’ arc (3’ flanking regulatory sequences of the arc5-I gene); RB, T-DNA right border
1141
(lower panel). C. CDR3 sequences of 13 N1rec-binding VHH candidates. The VHH CDR3 is
1142
highlighted by a red dotted box. The colored histogram denotes the homology between the
1143
sequences: red, 100%; orange, 60–80%; green, 40–60 %; light blue, 20–40 %; dark blue,
15448
-
>32896
-
>15448
-
>32896
-
>32896
123 -
0.157 (+/0.206) 0.69 (+/0.231) >15448 1.31 (+/0.605) 1.49 (+/0.746)
2708
7.6 (+/- 3.0)
240
52.2 (+/36.8)
543
14.5 (+/- 11.1)
58
>707.5 >32896 89.4 (+/7.8)
c
Fold
4295 (+/2835.4) 3492 (+/2967.6)
-
>32896
-
>32896
-
24.6 (+/31.9) 23.6 (+/3.8) >32896 24.2 (+/3.7)
174 148
-
>32896
-
324
23.08
79
251
28.14
30
>17083
-
27.03
129
>7900
-
>5260
-
>5260
-
>5260
-
>7900 586.1 (+/120.5)
-
>5260 15030 (+/10039)
-
>5260
-
>5260
-
-
>243000
-
73000
-
-
-
b
NIBRG-23
71
177
Mean inhibitory concentration (IC50) from three independent neuraminidase inhibition
assays using 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and 160 ng of N1rec. b
Mean plaque inhibitory concentration (IC50) from two independent plaque assays performed
in duplicate, in which the VHH concentration reduced the plaque size by 50% compared with mock VHH. c
Fold increase inhibitory potency of the N1-VHH bivalent format compared to its monovalent
format. d
Monomeric VHH against seed storage albumin.
e
Bivalent VHH against bacterial beta lactamase.
f
Coronavirus GP4 protein fused to mouse IgG2a Fc
Table 3. Endotoxin levels in the purified VHH preparations VHH N1-3-VHHm N1-5-VHHm N1-7-VHHm SA-VHHm N1-3-VHHb N1-5-VHHb BL-VHHb N1-3-VHH-Fc N1-5-VHH-Fc N1-7-VHH-Fc GP4-Fc PBS
a
LPS content (EU/ml)a 0.094 (+/- 0.007) 0.096 (+/- 0.017) 0.095 (+/- 0.012) 0.094 (+/- 0.008) 0.092 (+/- 0.005) 0.093 (+/- 0.007) 0.107 (+/- 0.024) 0.100 (+/- 0.020) 0.103 (+/- 0.019) 0.076 (+/- 0.011) 0.100 (+/- 0.016) 0.07 (+/- 0.015)
Endotoxin levels in the purified E. coli (VHHm/b) and A. thaliana (VHH-Fc and GP4-Fc)
derived proteins was determined using Limulus amebocyte lysate-based chromogenic test and calculated relative to an endotoxin standard.
1
Single domain antibodies targeting neuraminidase protect against an H5N1 influenza
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virus challenge
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Francisco Miguel Cardoso1,2 , Lorena Itatí Ibañez1,2$, Silvie Van den Hoecke1,2, Sarah De
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Baets1,2, Anouk Smet1,2, Kenny Roose1,2, Bert Schepens1,2, Francis J. Descamps1,2#, Walter
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Fiers1,2, Serge Muyldermans3,6, Ann Depicker4,5, Xavier Saelens1,2
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Belgium
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Brussels, Belgium
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$
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VIB Inflammation Research Center, Technologiepark 927, Ghent, Belgium Department for Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent,
Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussels, Belgium Department of Plant Systems Biology, VIB, Technologiepark 927, Ghent, Belgium Department of Biotechnology and Bioinformatics, Technologiepark 927, Ghent, Belgium Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050
Current address: ICT-Milstein – CONICET, Ciudad Autónoma de Buenos Aires, Argentina Current address: Ablynx NV, Ghent, Belgium
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Address correspondence to: Ann Depicker, [email protected], or Xavier Saelens,
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[email protected]
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Running Title: NA single domain antibodies protect against H5N1
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Word Count for abstract: 220
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Word Count for text: 9790
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ABSTRACT
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Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We
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isolated a set of H5N1 NA-specific single domain antibodies (N1-VHHm) and evaluated their in
26
vitro and in vivo antiviral potential. Two of them inhibited NA activity and in vitro replication of
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clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two
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methods. First we made N1-VHHb by genetically joining two N1-VHHm moieties by a flexible
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linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm
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moiety the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1
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of both bivalent N1-VHHb formats was 30- to 240-fold higher than their monovalent
32
counterparts, with IC50 values in the low nanomolar range. Moreover, single-dose prophylactic
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treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal
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challenge with H5N1 virus, including an Oseltamivir-resistant H5N1 variant. Surprisingly, also an
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N1-VHH-Fc fusion without in vitro NA inhibitory or antiviral activity protected mice against H5N1
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challenge. Virus escape selection experiments indicated that one amino acid residue close to
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the catalytic site is required for N1-VHHm binding. We conclude that single domain antibodies
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directed against influenza NA protect against H5N1 virus infection, and when engineered with a
39
conventional Fc domain, they can do so in the absence of detectable NA inhibitory activity.
40
IMPORTANCE
41
Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of bird flu caused by these
42
viruses occur in many parts of the world, are associated with tremendous economic loss and
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these viruses can cause very severe disease in human. In such cases, small molecule inhibitors
44
of the viral neuraminidase are among the few treatment options for patients. However treatment
45
with such drugs often results in the emergence of resistant viruses. Here we show that single
46
domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro
3
47
and can protect laboratory mice against challenge with an H5N1 virus, including an oseltamivir-
48
resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain, can protect
49
in vivo even in the absence of NA inhibitory activity. Thus, NA of influenza virus can be
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effectively targeted by single domain antibody fragments, which are amenable to further
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engineering.
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INTRODUCTION
53
Zoonotic influenza A virus infections are a persistent threat due to their pandemic potential. In
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particular, highly pathogenic avian influenza viruses (HPAIV) of the H5N1, H7N1 and H7N7
55
subtypes occasionally cross the species barrier between domesticated birds and human. These
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viruses could become transmissible between humans through reassortment with circulating
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swine or human influenza viruses or by gradually accumulating mutations (1, 2). In the last
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decade, zoonotic outbreaks had a major effect on public health. HPAIV H5N1 (3), the swine flu
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(H1N1) outbreak in 2009 (4), and more recently, human infections with H7N9 in South Asia (5)
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illustrate our poor preparedness for pandemic influenza (6). HPAIV H5N1 infection in humans
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has a confirmed case fatality rate of approximately 60%. The high pathogenicity of HPAIV H5N1
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in humans can be attributed to a high replication rate and a broad cellular tropism that can lead
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to systemic virus spread. In addition, deregulated induction of proinflammatory cytokines and
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chemokines (“cytokine storm”) is associated with severe HPAIV H5N1 infections, and can result
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in a disproportionate immunological response (7).
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Influenza neuraminidase (NA) is a homotetrameric type II membrane glycoprotein with sialidase
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activity. The NA catalytic site is located at the top of each monomer, opposite to the tetramer
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interface. NA plays an essential role in the spread of influenza viruses by cleaving sialic acids
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from the host cell receptors and from virions. NA activity also contributes to virus entry by
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cleaving decoy receptors present in mucins that line the layer of respiratory epithelial cells (8).
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Immunologically, NA is the second major humoral antigenic determinant (after HA) and is
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subject to antigenic drift and occasional shift. In addition, experimental influenza vaccines
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supplemented with NA have improved efficacy (9-11). NA is also a co-determinant of influenza
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A virus (IAV) pathogenicity (12-14) and is involved in limiting IAV superinfections and
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reassortment (15). Decreased NA activity has been correlated with H5N1 adaptation to human
5
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airway epithelium (16) and antibodies against NA contribute to protection against H5N1 virus
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challenge in a mouse model (17).
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Hemagglutinin (HA), the other major antigen, and NA cooperate in a tightly controlled way. For
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example, the fitness of mutant IAV virus lacking NA activity can be rescued by selection of HA
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mutants with decreased affinity for receptors containing sialic acid (18-20). These data
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demonstrate the importance of NA during IAV infection, so targeting NA is a rational strategy.
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Indeed, three licensed influenza antivirals, Oseltamivir, Zanamivir and Peramivir, target NA.
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Influenza viruses that are resistant to Oseltamivir frequently emerge in humans. In addition, NA-
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specific antibodies protect mice, and serum anti-NA antibodies are associated with resistance to
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influenza A virus challenge in humans (21).
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Effective prevention and treatment strategies are needed to control H5N1 infections and single-
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domain antibody-based technology have been described as promising antivirals (22). Naturally
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occurring single heavy chain antibodies have been found in sharks and camelids (23, 24).
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Unlike conventional antibodies that are typically composed of a light and a heavy polypeptide
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chain that together determine the epitope specificity, these natural single-chain antibodies are
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composed of heavy chains only, and hence their epitope specificity is confined in a single
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domain (so called VHH, Variable domain of Heavy chain). Single-domain antibody fragments
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derived from camelids are highly specific and robust, easy to produce and flexible, and hence
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they are used as novel therapeutics for many diseases (25, 26). VHHs usually have extended
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antibody loop structures and can interact with cognate antigens with affinities matching the best
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affinities of conventional antibodies (27-29). In addition, VHHs that potently inhibit enzymes
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have been described (30, 31), and they have been used to stabilize membrane proteins in order
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to determine crystal structure (32). The VHH tends to target concave-shaped epitopes, in
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contrast to the planar epitopes that are typically recognized by “classical” light and heavy chain
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antibodies (33). In addition, several examples of VHHs with potent antiviral activity have been
6
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reported (34-36). We previously reported on an HA-specific H5N1 neutralizing VHH (35) and
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H5N2 neutralizing VHH were reported more recently (37). Moreover, a VHH directed against the
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influenza M2 protein inhibited replication of amantadine-sensitive and amantadine-resistant
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viruses and protected mice against lethal influenza challenge (38). Recently, Harmsen et al.
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described a panel of NA-binding VHHs, some of which displayed cross-subtype NA-binding, but
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the antiviral potential of these VHHs was not evaluated (39).
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Here, we report the isolation and characterization of a set of H5N1 NA-binding VHHs (N1-
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VHHm) of which some have potent NA inhibitory activity and in vitro and in vivo antiviral
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activities. We also describe the production of bivalent in tandem N1-VHHm formats in
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Escherichia coli and N1-VHHm proteins fused to a mouse IgG2a Fc domain in seeds of
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transgenic Arabidopsis plants. Further, we compared the in vitro antiviral activity against H5N1
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clade 1 (Oseltamivir-sensitive and -resistant strains) and clade 2 viruses, and we evaluated the
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N1-VHHm based prophylactic protection of mice against a potentially lethal infection with H5N1
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virus.
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MATERIALS AND METHODS
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Influenza viruses
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H5N1 IAV strains NIBRG-14 and NIBRG-23 were obtained from the UK National Institute for
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Biological Standards and Control, a center of the Health Protection Agency. NIBRG-14 and
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NIBRG-23 are 2:6 reverse genetics–derived reassortants with NA and HA (lacking the polybasic
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cleavage site) segments derived from A/Vietnam/1194/2004 (H5N1) and A\turkey\Turkey\2005
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(H5N1), respectively, and the other six segments from A/PR/8/34 (H1N1) viruses. The H5N1
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H274Y virus described here is a 1:1:6 reverse genetics-derived reassortant, with NA derived
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from A/crested eagle/Belgium/01/2004 (40) carrying the H274Y mutation introduced by site-
124
specific mutagenesis, HA from NIBRG-14, and the remaining six genome segments from
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A/PR/8/34 (41). This virus was rescued by transfection of co-cultured HEK-293T and MDCK
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cells. The supernatant from these cells was used for end point dilution to obtain a clonal H5N1
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H274Y virus sample that was subsequently amplified on MDCK cells, pelleted from the cell
128
supernatant, and mouse adapted by serial passage in BALB/c mice. All HA segments of these
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H5N1 viruses lack the coding information for the polybasic cleavage site. Following adaptation
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to BALB/c mice, the HA and NA-coding regions of the mouse-adapted NIBRG-14 (NIBRG-14
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ma) and H5N1 H274Y (H5N1 H274Y ma) were sequenced and found to be identical to those of
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the parental viruses. pH1N1 (kindly provided by Dr. Bernard Brochier, Scientific Institute of
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Public Health, Brussels, Belgium) is derived from a clinical isolate of the pH1N1 virus of 2009
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and was adapted to mice by serial passages (42). A/New Caledonia/20/99 was kindly provided
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by Dr. Alan Hay (MRC National Institute for Medical Research, Mill Hill, London, UK). The
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median tissue culture infectious dose (TCID50) and median lethal dose (LD50) of NIBRG-14 ma
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and H5N1 H274Y ma viruses were calculated by the method of Reed and Muench (43). All the
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H5N1 and pH1N1 experiments described above were performed in BSL-2+ rooms.
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139
Baculovirus-based production of recombinant soluble tetrameric neuraminidase
140
A soluble and enzymatically active tetrameric NA (N1rec) derived from A/crested
141
eagle/Belgium/01/2004 (H5N1) was produced by proprietary technology (WO 2002/074795).
142
Briefly, the N1rec expression cassette consisted of the secretion signal of IAV HA (ssHA, 16
143
residues), a tetramerizing variant of the leucine zipper domain of the Saccharomyces cerevisiae
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GCN4 transcription factor (tGCN4, 32 residues) (44, 45), and the extracellular part of NA
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derived from A/crested eagle/Belgium/01/2004 (H5N1) (amino acid residues 53–449) (40) (Fig.
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1A). This N1rec expression cassette was cloned into the pAcMP2 baculovirus transfer vector to
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produce pAcMP2n1rec. Recombinant baculovirus was generated by co-transfection of
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pAcMP2n1rec and linearized DNA of Autografa californica Nuclear Polyhedrosis Virus (AcNPV)
149
(BaculoGold, BD Biosciences) into Sf9 insect cells (derived from Spodoptera frugiperda), using
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the ESCORT IV Transfection Reagent (Sigma-Aldrich) to generate N1rec encoding recombinant
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baculovirus. Sf9 cells infected with AcNPVN1rec were incubated at 28°C in rolling bottles in TC-
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100 medium (Invitrogen), supplemented with 10% FCS and penicillin + streptomycin. Four days
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after infection, the supernatant was harvested and clarified by centrifugation (1 h at 50,000 g).
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Recombinant neuraminidase purification
155
All purification steps were carried out at 4°C. Two parts (v:v) of n-butanol were added to three
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parts of cleared AcNPVN1rec-infected Sf9 cell supernatant and the mixture was shaken
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vigorously for 5 min. The aqueous phase, containing soluble N1rec, was separated from the
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organic phase, diluted 2.5 times in 5 mM KH2PO4 pH 6.6 and passed through a 0.22-µm filter.
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The diluted aqueous phase was subsequently applied to an XK26\70 column (GE Healthcare)
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packed with HA Ultrogel Hydroxyapatite Chromatography Sorbent (Pall), and eluted with a
161
gradient of 5-400 mM KH2PO4 pH 6.6 in 4% butanol. Eluted fractions with high NA activity were
162
pooled and loaded onto a Blue Sepharose (Sigma-Aldrich) column, equilibrated with 50 mM
9
163
MES pH 6.6 containing 5% glycerol and 8 mM CaCl2. Bound proteins were eluted with 50 mM
164
MES pH 6.6 containing 5% glycerol, 8 mM CaCl2 and 1.5 M NaCl. Fractions with NA activity
165
were loaded on a HiLoad 16/60 Superdex 200 (GE Healthcare) gel filtration column pre-
166
equilibrated with 50 mM MES pH 6.6 with 5 % glycerol and 8 mM CaCl2, 150 mM NaCl. Peak
167
fractions containing NA activity were pooled, aliquoted, and stored at -80ºC until used. All the
168
chromatography steps were performed using an Akta purification station (GE Healthcare).
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Neuraminidase activity assays
170
NA activity was quantified by measuring the rate of cleavage of the fluorogenic substrate 4-
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MUNANA (2’-4-Methylumbelliferyl-α-D-N-acetylneuraminic acid, sodium salt hydrate (Sigma-
172
Aldrich) into 4-methylumbelliferone. The NA activity reaction was performed in 200 mM NaAc, 2
173
mM CaCl2 with 1 % butanol and 1 mM 4-MUNANA and measured in a kinetic mode, with
174
excitation at 365 nm and emission at 450 nm in an Optima Fluorostar. A standard curve of
175
increasing concentrations of soluble 4-methylumbelliferone was included to correlate the
176
fluorescence intensity with the molar amount of 4-methylumbelliferone. One NA activity unit is
177
defined as the activity needed to generate 1 nmol of 4-methylumbelliferone per min.
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Fetuin (5 µg/ml; Sigma-Aldrich) was coated on Nunc 96 well plates overnight at 4°C. Excess
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fetuin was washed away with PBS, and IAV dilutions (diluted in PBS with 1 mM CaCl2 and 0.5
180
mM MgCl2), with or without added single domain antibodies, were added and incubated 1 h at
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37°C. The amount of desialylated fetuin was measured by colorimetry to determine binding of
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horseradish peroxidase (HRP) coupled peanut agglutinin (PNA, Sigma-Aldrich). The plates
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were washed three times with PBS + 0.1% Tween 20 and then incubated with 50 µl PNA-HRP
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(2.5 µg/ml in PBS + 0.05% Tween 20) at room temperature. Then the plates were washed three
185
times with PBS, after which 50 µl of TMB substrate (Pharmigen BD) was added and absorbance
186
was measured at 450 nm with a reference at 650 nm. In the Accelerated Viral Inhibition Assay
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(AVINA) (46), IAV dilutions containing the indicated N1-VHHm concentrations were transferred
10
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to a black 96-well plate and 75 μl of 20 μM MUNANA was added and incubated 1 h at 37°C.
189
Then, 100 μl stop solution (0.1 M glycine, pH 10.7, 25% ethanol) was added to each well, and
190
fluorescence was determined. For the AVINA and fetuin substrate assays, we used the
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following amounts of IAV: 7 x 105 pfu of NIBRG-14 ma, 1 x 104 pfu of H5N1 H274Y ma, and 3 x
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104 plaque forming units (pfu) of pH1N1.
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Camelid immunization, phage library construction, and panning
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An alpaca (Vicugna pacos) was injected subcutaneously with 125 µg of N1rec in the presence
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of incomplete Freund’s adjuvant. Injections were once a week for five weeks. On day 39, anti-
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coagulated blood was collected, lymphocytes were isolated using a UNI-SEP density gradient
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separation kit (NOVAmed), and total RNA was extracted. cDNA was prepared using oligo (dT)
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primers,
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(GTCCTGGCTGCTCTTCTACAAGG)
200
(GGTACGTGCTGTTGAACTGTTCC). PstI and NotI restriction sites were inserted into the
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amplified sequences using the primers A6E (GATGTGCAGCTGCAGGAGTCTGGRGGAGG)
202
and 38 (GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT). The resulting PCR product
203
(550 bp) and the pHEN4 vector were digested with PstI and NotI, ligated (47), and transformed
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into electrocompetent TG1 E. coli cells. Transformants were grown in 2xTY medium,
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(supplemented with 100 µg/ml ampicillin and 1% glucose) and infected with helper phage
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M13K07. The resulting VHH phage display library was panned four times using solid-phase
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coated N1rec.
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Production and purification of monovalent neuraminidase-binding VHH (N1-VHHm)
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The VHH coding sequence of the selected N1rec-binding phages (n = 13) was amplified by
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PCR with the primers A6E and 38. The N1rec-binding VHH genes obtained by PCR and the
and
the
VH
and
VHH
genes
were and
amplified
with
primer
primer
CALL001 CALL002
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pHEN6c vector were digested with PstI and BstEII, ligated, and transformed into E. coli WK6
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strain. In the pHEN6c vector, the coding sequence for the N1rec-binding VHH candidates were
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cloned downstream of an N-terminal PelB secretion signal sequence and a carboxyterminal
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hexahistidine tag was introduced (Fig. 1B)(48). For production and purification of soluble VHH,
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pHEN6c-transformed WK6 cells were grown in TB medium supplemented with 100 µg/ml
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ampicillin, 2 mM CaCl2 and 0.1% glucose. After 16 hours, VHH production was induced with 1
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mM of isopropyl β-D-1-thiogalactopyranoside, and the periplasmic fraction was extracted by
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osmotic shock using 0.2 M Tris pH 8.0, 0.5 mM EDTA and 0.5 M sucrose. Periplasmic extracts
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were centrifuged at 8000 rpm and 4°C and the supernatant was applied to a His Select Nickel
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Affinity gel (Sigma-Aldrich). After loading, the column was washed with PBS, and VHHs were
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eluted with 0.5 M imidazole and dialyzed against PBS at 4°C. Monovalent N1rec-binding VHH
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(N1-VHHm) was concentrated with a Vivaspin 5000-kDa cutoff filter (Vivascience). Total protein
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concentration was determined with the BCA protein Assay kit (Thermo Scientific) and endotoxin
224
levels with a commercial Limulus Amebocyte Lysate-based assay (Toxinsensor Chromogenic
225
LAL Endotoxin Assay Kit, Genscript Corp.). We obtained 13 recombinant N1 NA-specific VHH
226
proteins, which were named N1-1-VHHm to N1-13-VHHm. An irrelevant monovalent VHH
227
against Arabidopsis thaliana albumin (SA-VHHm) was used as a negative control (49). The N1-
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VHHm sequences were aligned using the MegAlign software (DNAstar) and the VHH CDR3
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electronegativity surface potential was predicted with the open source software ESyPred3D and
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visualized with DeepView (50)(Fig. 1C and 1D).
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ELISA assays
232
N1rec or M2e-tGCN4 (44) at 10 µg/ml was used to coat O/N in 96-well Maxisorp (Nunc) plates
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using 100 µl per well. Blocking was performed overnight with 5% skim milk in PBS. Dilution
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series of the 13 N1-VHHm candidates (from 10 µM - 0.1 nM) were incubated for 2 h at 37°C,
12
235
and excess VHH was removed by three washes with PBS containing 0.05 % Tween 20. Binding
236
of the N1-VHHm to N1rec tGCN4 or NA globular head moieties was assessed by a mouse anti-
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His-tag antibody fused with horse radish peroxidase (HRP) (1:5000). After three washes with
238
PBS, 100 µl of Tetramethylbenzidine (TMB) substrate (Pharmigen BD) was added, and
239
absorbance was measured at 450 nm.
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N1-VHHm surface plasmon resonance analysis
241
The affinity of monovalent N1-3-VHHm, N1-5-VHHm and N1-7-VHHm for N1rec was
242
determined by surface plasmon resonance (SPR) on a Biacore 3000 platform (GE Healthcare).
243
N1rec antigen was first immobilized (2000 resonance units) into a Series S sensor chip CM5
244
(GE Healthcare) by chemical -NH2 coupling, in 10 mM NaAc pH 4.5. N1rec loaded channels
245
were regenerated with 0.02% SDS. Each monovalent N1-VHHm was diluted in HBS buffer (0.01
246
M HEPES, 0.15 M NaCl, 0.005% Tween 20, pH 6.4) to obtain a gradient ranging from 1.95 to
247
1000 nM. The different N1-VHHs were injected over the N1rec coated chip to record their
248
binding kinetics. The Biacore T00 Evaluation Software was used to calculate the association
249
rate constants (Kon), dissociation rate constants (Koff) and the equilibrium dissociation constants
250
(KD = Koff/Kon).
251
Construction of bacteria-produced bivalent N1-VHH (N1-VHHb)
252
The coding information for N1-3-VHHm and N1-5-VHHm was amplified with primers MH
253
(CATGCCATGGGAGCTTTGGGAGCTTTGGAGCTGGGGGTCTTCGCTGTGGTGCGCTGAGG
254
AGACGGTGACCTGGGT)
255
(CATGCCATGATCCGCGGCCCAGCCGGCCATGGCTGATGTGCAGCTGGTGGAGTCT)
256
introduce an NcoI restriction enzyme site at both ends of the amplified fragment. The MH primer
257
also introduced the hinge sequence of llama γ2c (AHHSEDPSSKAPKAPMA) (51) to separate
258
the two N1-VHHm domains. PCR amplicons of N1-3-VHHm and N1-5-VHHm were cloned into
and
A4short to
13
259
pHEN6c plasmid containing the n1-3-vhh or the n1-5-vhh gene in order to generate homo-
260
bivalent N1-3-VHHb and N1-5-VHHb expression constructs, which also contained a carboxy-
261
terminal hexahistidine tag (Fig. 1B). These bivalent constructs were expressed and purified from
262
the periplasmic fraction of E. coli as outlined above. We used an irrelevant bivalent in tandem
263
fused VHH specific for bacterial beta lactamase (BL-VHHb) as a negative control (52) (kindly
264
provided by Dr. Trong Nguyen-Duc, VUB, Brussels). Endotoxin levels of the bivalent VHH
265
preparations were determined as above.
266
Production of bivalent N1-VHHb fused to the mouse IgG2 Fc fragment (N1-VHH-Fc) in
267
seeds of stably transformed Arabidopsis thaliana lines
268
The three different n1-vhh-fc sequences coding for N1-3VHHm, N15-VHHm and N1-7-VHHm
269
fused at the aminoterminal region with the 2S2 signal peptide and at the carboxyterminal region
270
with the Fc region of mouse IgG2a and the KDEL retention signal were synthesized
271
commercially (GenScript), and cloned into a PphasGW binary T-DNA vector, resulting in the
272
PphasGWn1-vhh-fc vector (Fig. 1B). The Coronavirus GP4 subunit protein fused to the same
273
mouse IgG2a Fc moiety (GP4-Fc, kindly provided by Robin Piron, VIB and Ghent University)
274
was used as an irrelevant control in the antiviral assays. Agrobacterium C58C1RifR (pMP90)
275
was transformed with PphasGWn1-3-vhh-fc, PphasGWn1-5-vhh-fc, PphasGWn1-7-vhh-fc or
276
PphasGWGP4-fc constructs.
277
Agrobacterium
278
PphasGWn1-5-vhh-fc, PphasGWn1-7-vhh-fc and PphasGWGP4-fc binary T-DNA vectors and
279
the resulting strains were used for Arabidopsis thaliana floral dip transformation (53, 54). T1 to
280
T3 generations were produced and screened for best N1-VHH-Fc expressers. For protein
281
purification, 1 g of transformed A. thaliana seeds was crushed in 25 ml of 50 mM Tris-HCl, pH
282
8.0, 200 mM NaCl, 5 mM EDTA, 0.1% (v/v) Tween 20 and Complete protease inhibitor tablets
C58C1RifR (pMP90)
was
transformed
with
the
PphasGWn1-3-vhh-fc,
14
283
(Roche). The seed extracts were clarified by centrifugation and applied to a protein G
284
sepharose column (GE Healthcare). N1-VHH-Fc proteins were eluted in 0.1 M glycine pH 3.0
285
and immediately neutralized with 1 M Tris-HCl, pH 9.0. The recovery was between 1 and 10 mg
286
of N1- VHH-Fc. Endotoxin levels of the VHH-Fc preparations were determined as above.
287
Plaque assays
288
Monolayers of MDCK cells were grown in DMEM supplemented with 10% fetal calf serum, 1%
289
penicillin + streptomycin and 1% glutamine and non-essential amino acids at 37°C in 5% CO2.
290
MDCK TMPRSS2 cells (kindly provided by Dr. Wolfgang Garten, Philipps-Universität Marburg,
291
Germany) were cultured in DMEM supplemented with the above as well as geneticin (0.3
292
mg/ml) and puromycin (2 µg/ml). Expression of TMPRSS2 was induced with doxycycline (0.5
293
µg/ml) (55). At 70% confluence, the MDCK cells were infected at a multiplicity of infection (moi)
294
of 10 with NIBRG-14 ma, NIBRG-23 or H5N1 H274Y ma. Samples containing monovalent N1-
295
3/5/7-VHHm, bivalent N1-3/5-VHHb, bivalent N1-3/5/7/GP4-VHH-Fc, Oseltamivir or control
296
medium were mixed with 0.8% Avicel RC-591™ as overlay (56). After 2 to 4 days of incubation,
297
the cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were permeabilized
298
with 20 mM glycine containing 0.5% Triton X-100 (v/v). After blocking with BSA, the cells were
299
incubated for 2 h with polyclonal α-NIBRG-14 (1:1000) or anti-M2e monoclonal antibody
300
(1:5000). After washing, α-mouse whole IgG-HRP was used to visualize plaques with the
301
substrate TrueBlue™ Peroxidase Substrate (KPL).
302
Mouse experiments
303
All mouse experiments were conducted according to national (Belgian Law 14/08/1986 and
304
22/12/2003, Belgian Royal Decree 06/04/2010) and European legislation (EU Directives
305
2010/63/EU, 86/609/EEG) on animal regulations. All experiments on mice were approved by the
306
ethics committee of Ghent University (permit number LA1400091) and efforts were made to
15
307
avoid or diminish suffering of the animals. Specific-pathogen–free female BALB/c mice, 7–9
308
weeks old, were purchased from Charles River (Germany) and used in all experiments. Mice
309
were housed in ventilated cages with high-efficiency particulate air filters in temperature-
310
controlled, air-conditioned facilities and had food and water ad libitum. Mice were anaesthetized
311
by intraperitoneal injection of xylazine (10 μg/g) and ketamine (100 μg/g) before intranasal
312
administration of N1-VHHm, N1-VHHb or N1-VHH-Fc. The N1-VHHm in any format were diluted
313
in endotoxin-free phosphate-buffered saline (PBS) with 1% (w/v) bovine serum albumin, and
314
applied intranasally in doses ranging from 0.25 to 5 mg/kg, as indicated in the figure legends.
315
Mice were challenged 4 or 24 h later, as indicated, with 6 LD50 (H5N1 H274Y ma) or 4 LD50
316
(NIBRG-14 ma) of virus (50 μl, divided equally between the nostrils). A 30% loss in body weight
317
was the end point at which sick mice were euthanized. On day 4 after challenge, lung
318
homogenates were prepared in PBS, cleared by centrifugation at 4°C, and used for virus
319
titration. Monolayers of MDCK cells were infected with 50 μl of serial 1:10 dilutions of the lung
320
homogenates in a 96-well plate in serum-free Dulbecco's modified Eagle medium (Invitrogen)
321
supplemented with L-glutamine, non-essential amino acids, penicillin and streptomycin. After 1
322
h, the inoculum was replaced by medium containing 2 μg/ml of L-(tosylamido-2-phenyl) ethyl
323
chloromethyl ketone–treated trypsin (Sigma-Aldrich). End-point virus titers were determined by
324
hemagglutination of chicken red blood cells and expressed as TCID50 per milliliter. IAV RNA
325
levels were determined by quantitative RT-PCR. RNA was isolated from 150 μl of cleared lung
326
homogenate using the Nucleospin RNA virus kit (Machery-Nagel). The relative amount of
327
NIBRG-14 ma genomic RNA was determined by preparing viral cDNA and performing
328
quantitative PCR with M-genomic segment primers (CGAAAGGAACAGCAGAGT and
329
CCAGCTCTATGCTGACAAAATG) and probe (GGATGCTG) (probe no. 89; Universal
330
ProbeLibrary, Roche) and the LightCycler 480 Real-Time PCR System (Roche).
16
331
Selection and characterization of H5N1 N1-VHHm escape mutant viruses
332
NIBRG-14 ma virus was serially diluted and used to infect MDCK cells in the presence of N1-3-
333
VHHm (4.2 µM) or N1-H5-VHHm (3.7 µM), a dose corresponding to ten-fold the IC50 (Table 2).
334
Escape mutants were screened by monitoring cytopathic effects. After 9 passages in the
335
presence of N1-3-VHHm or N1-5-VHHm, escape viruses were plaque purified by growth on
336
MDCK cells overlaid with 0.6% low-melting agarose in the presence of N1-3-VHHm or N1-5-
337
VHHm. Escape virus isolates were amplified on MDCK cells, still in the presence of a ten-fold
338
IC50 of N1-3-VHHm or N1-5-VHHm. Total RNA was extracted from the supernatant and used to
339
clone the cDNA corresponding to the NA viral RNA segment (57). Deduced amino acid
340
substitutions in the NA sequence of escape viruses were modeled with PyMol (Delano
341
Scientific,
342
A/Vietnam/1194/2004 (PDB code 2HTY). N1-VHH binding assays were performed as described
343
(35). Briefly, 4-8 HA unit of wild type or plaque purified candidate escape viruses were used to
344
coat ELISA plates. After blocking, wells were incubated with different concentrations of N1-3-
345
VHHm, N1-3-VHHb, N1-5-VHHm or N1-5-VHHb. Binding of N1-VHHs was revealed by
346
incubation with an anti-His tag monoclonal antibody, followed by a secondary sheep anti-mouse
347
IgG antibody conjugated to HRP and visualized by addition of TMB substrate and absorbance
348
was measured at 450/655 nm.
349
Statistical analysis
350
Graphpad software (Graphpad Prism, version 6) was used for statistical analysis. For
351
comparing two groups in the fetuin and AVINA assays, t test was used. Differences between
352
mouse groups were tested using two-way ANOVA. When this test demonstrated a significant
353
difference between groups (P < 0.05), t tests were used to compare two groups on each day.
354
Kaplan-Meier survival curves were plotted and differences in survival were analyzed by the
355
Mantel-Cox log-rank test.
http://www.pymol.org)
using
the
H5N1
NA
structure
derived
from
17
356
RESULTS
357
Production of recombinant tetrameric neuraminidase
358
A Baculovirus Expression Vector System was used to produce recombinant soluble tetrameric
359
and enzymatically active H5N1 NA derived from A/crested eagle/Belgium/01/2004 (N1rec). The
360
N1rec coding sequence was cloned into a pAcMP2 expression vector (pAcMP2n1rec), under
361
the control of the AcNPV basic protein promoter that is active in the late phase of the
362
Baculovirus infection cycle (Fig. 1A). Infection of Spodoptera frugiperda (Sf9) cells with the
363
recombinant Baculovirus AcNPVN1rec resulted in sialidase activity in the cell supernatant,
364
suggesting that the N1rec product was soluble and enzymatically active NA (Fig. 1A). N1rec
365
protein was purified from the supernatant of infected Sf9 cells by three successive
366
chromatography steps, including size exclusion chromatography to collect tetrameric N1rec (see
367
Materials and Methods). Using MUNANA as a substrate, we calculated that purified N1rec had
368
a specific activity of 56,851 NA units/mg.
369
Immunization and VHH phage library construction
370
N1rec was used as an antigen for the generation and selection of NA-specific VHH. From an
371
immunized alpaca, we obtained a VHH phage library of 2 X 108 independent transformants,
372
57% of which had a cDNA insert of the correct size (data not shown). Seventy-eight positive
373
clones were selected, 13 of which had unique VHH sequences. The complementarity
374
determining region 3 (CDR3) had the most variable amino acid sequence among the 13 N1rec-
375
binding VHHs (N1-VHHm) (Fig. 1C). In silico structure prediction of the monomeric N1-VHHm
376
candidates showed that the CDR3 of N1-1-VHHm, N1-3-VHHm, N1-4-VHHm, N1-5-VHHm and
377
N1-6-VHHm had an electronegative protruding paratope (Fig. 1D).
378
Production and characterization of soluble monomeric N1-VHHm
18
379
The binding of the N1-VHHm proteins to N1rec was assessed by ELISA and compared with
380
binding to the recombinant M2e-tGCN4 protein, which like N1rec contains the same
381
tetramerizing leucine zipper (Table 1 and Fig. 1E). Except for N1-11-VHHm and N1-13-VHHm,
382
none of the N1-VHHm bound substantially to recombinant M2e-tGCN4 protein. We next used
383
the MUNANA substrate to assess the ability of the N1-VHHm candidates to inhibit the
384
enzymatic activity of N1rec. Four candidates, N1-1-VHHm, N1-3-VHHm, N1-5-VHHm and N1-6-
385
VHHm, inhibited the N1rec catalytic activity (Table 1). N1-3-VHHm and N1-5-VHHm inhibited
386
N1rec in the sub-micromolar range (Table 2), and together with N1-7-VHHm, they were
387
analyzed by SPR to determine their affinity for immobilized N1rec. Both N1-3-VHHm and N1-5-
388
VHHm had a high affinity for N1rec, with an equilibrium dissociation constant (KD) in the low
389
nanomolar (N1-5-VHHm) to high picomolar (N1-3-VHHm) range. N1-7-VHHm showed
390
approximately 10- to 100-fold lower affinity for N1rec than N1-5-VHHm and N1-3-VHHm,
391
respectively (Table 1). These binding affinities of N1-3-VHHm and N1-5-VHHm for N1rec
392
resemble the affinities reported for a monomeric VHH that inhibits the activity of lysozyme (33).
393
Competitive SPR analysis also showed that prior binding of N1-3-VHHm to N1rec abolished
394
subsequent binding of N1-1-VHHm, N1-5-VHHm and N1-6-VHHm. Conversely, N1-7-VHHm
395
binding to N1rec was not affected by prior incubation of N1rec with N1-3-VHHm or N1-5-VHHm
396
(Fig. 1F). This suggests that N1-3-VHHm and N1-5-VHHm bind an overlapping epitope within
397
N1rec, or that binding of N1-3-VHHm sterically hinders binding of N1-5-VHHm, while N1-7-
398
VHHm binds a different epitope.
399
We next analyzed the antiviral potential of N1-3-VHHm and N1-5-VHHm using the H5N1 strain
400
NIBRG-14 ma. The amino acid sequences of the NA of NIBRG-14 ma (derived from
401
A/Vietnam/1194/2004) and N1rec (derived from A/crested eagle/Belgium/01/2004) are highly
402
homologous, with only 13 amino acid differences, none of which map in or close to the NA
403
catalytic site. In the presence of N1-3-VHHm or N1-5-VHHm, the size of NIBRG-14 ma plaques
19
404
was reduced in a concentration dependent manner, and with IC50 values in low nanomolar
405
range (Table 2). In contrast, N1-7-VHHm did not affect the size or number of NIBRG-14 ma
406
plaques (Table 2). These results indicate that the in vitro antiviral potential of NA-specific VHH
407
proteins depends on their NA-inhibitory activity.
408
Bivalent N1-VHHb formats have enhanced NA inhibitory and antiviral potential
409
It has been reported that generation of multivalent formats of VHHs increases their functional
410
affinity by introducing avidity (34). For example, the introduction of avidity drastically decreased
411
the dissociation constant (Koff) of the VHH molecules directed against lysozyme, and
412
significantly improved their enzyme inhibitory activity over their monovalent format (51). To
413
increase the avidity of the N1-VHHm, we compared two different bivalent formats. In one
414
approach, we used the llama IgG2c hinge (17 amino acid residues) as a flexible linker to fuse
415
two identical N1-VHHm gene moieties in tandem, resulting in the bivalent n1-3-vhhb and n1-5-
416
vhhb expression elements (Fig. 1B and 2A). Despite repeated attempts, we could not generate
417
an N1-7-VHHb expression plasmid. The corresponding proteins were expressed and purified
418
from E. coli and their in vitro N1-VHHb NA-inhibitory and antiviral activities were compared with
419
those of their monovalent counterparts. N1rec inhibition by N1-3-VHHb and N1-5-VHHb was
420
2000- and 500-fold stronger than the corresponding N1-3-VHHm and N1-5-VHHm formats,
421
respectively (Table 2). Surprisingly, in a plaque assay using MDCK cells infected with NIBRG-
422
14 ma virus, both N1-3-VHHb and N1-5-VHHb seemingly had antiviral activities that were
423
comparable to the activities of their monovalent counterparts (data not shown). In this assay,
424
exogenous trypsin is used to facilitate maturation of HA in newly produced virions to allow
425
multicycle replication of the recombinant NIBRG-14 ma virus. We noticed that the llama IgG2c
426
hinge linker used to generate bivalent VHH formats was sensitive to trypsin cleavage (Fig. 2A).
427
To circumvent the use of exogenously added trypsin, we took advantage of TMPRSS2 MDCK
428
cells, which are transformed with the doxycycline-inducible serine protease TMPRSS2 and
20
429
allow multicycle replication of IAV in the absence of exogenous trypsin (55). Using monolayers
430
of TMPRSS2 MDCK cells for infection with NIBRG-14 ma, the antiviral effect of N1-3-VHHb and
431
N1-5-VHHb was increased 240- and 58- fold, respectively, compared with their monovalent
432
formats (Table 2). These results obtained with N1-VHHb indicate that there is a significantly
433
enhanced NA-inhibitory and antiviral activity of both bivalent N1-VHHb compared with the
434
monovalent N1-VHHm formats.
435
We next engineered N1-VHH variants fused to a mouse IgG2a-derived Fc domain as an
436
alternative bivalent format (N1-Vhh-Fc). To produce these more complex proteins, we used a
437
robust plant seed-based expression system with reported high yield of recombinant antibodies
438
(58, 59). For this, the n1-3-vhh, n1-5-vhh and the n1-7-vhh genes were fused to the sequence
439
encoding the hinge and Fc tail of mouse IgG2a (n1-vhh-fc). These n1-vhh-fc constructs were
440
cloned into the binary vector Pphas as a T-DNA expression cassette (Fig. 1B and 2B).
441
Subsequently, transgenic Arabidopsis thaliana plants were generated by Agrobacterium-
442
mediated floral dip transformation. Seed extracts of segregating T3 Arabidopsis transformants
443
were screened by ELISA using antibodies against the Fc part for the best expressers of the
444
recombinant dimeric N1-VHH-Fc proteins. N1-3-VHH-Fc and N1-5-VHH-Fc accumulated to high
445
levels in several transformants, and levels of 16 % of total soluble seed protein were found. On
446
the contrary, N1-7-VHH-Fc did not accumulate well and the best expressors contained about
447
1% VHH-Fc of total soluble seed protein (data not shown). A good correlation was observed
448
between the ELISA result and NA inhibitory activity in crude seed extracts from different T3
449
transformants (data not shown). N1-3-VHH-Fc, N1-5-VHH-Fc and N1-7-VHH-Fc were purified
450
from seed extracts by protein G affinity chromatography. Reducing SDS-PAGE and Coomassie
451
staining showed that the N1-VHH-Fc monomers migrated at approximately 42 kDa (Fig. 2B).
452
N1-3-VHH-Fc and N1-5-VHH-Fc had stronger N1rec inhibition capacity and in vitro antiviral
453
activity than their monovalent N1-3-VHHm and N1-5-VHHm counterparts (Table 2). Bivalent N1-
21
454
7-VHH-Fc bound to N1rec in ELISA assays (data not shown), but it failed to inhibit N1rec and
455
the H5N1 viruses in vitro. Taken together, these results indicate that the in vitro NA inhibition
456
and antiviral activity against NIBRG-14 ma virus is enhanced at least 30-fold by using bivalent
457
N1-VHHb and N1-VHH-Fc formats, compared with the monovalent N1-VHHm domain.
458
In vitro antiviral activity of N1-VHH against H5N1 clade 2.2, H5N1 Oseltamivir-resistant
459
and pandemic H1N1 2009 virus
460
Like NA from NIBRG-14, N1rec NA (derived from A/crested eagle/Belgium/01/2004) belongs to
461
the clade 1 of the H5N1 NAs. To assess the potential cross reactivity of the NA activity inhibition
462
(NAI) of the isolated VHHs in the three available formats (N1-VHHm, N1-VHHb and N1-VHH-
463
Fc), we focused first on NIBRG-23 NA (derived from A/turkey/Turkey/01/2005), a clade 2.2
464
H5N1 virus (60). Although there are only a few laboratory-confirmed human infections with
465
H5N1, it appears that clade 2 H5N1 viruses are responsible for most of these zoonotic
466
infections with HPAIV (61). N1-3-VHHm and N1-5-VHHm reduced in vitro growth of NIBRG-23
467
virus with an IC50 in the low micromolar range (Table 2). N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc
468
and N1-5-VHH-Fc displayed about 150-fold higher in vitro antiviral activity compared to the
469
corresponding monovalent N1-VHHm against this clade 2 virus, as judged by a plaque size
470
reduction assay. Based on these findings, we conclude that the two NA-inhibitory VHHs inhibit
471
in vitro replication of representative H5N1 IAV from clades 1 and 2 with a comparable efficiency,
472
suggesting that they target a common epitope shared by the NA of these viruses.
473
Oseltamivir-resistant IAV frequently emerges and spreads in the human population. Several
474
mutations have been reported to contribute to Oseltamivir-resistance, but among them the
475
H274Y mutation (N2 numbering) is the most common found in Oseltamivir-resistant viruses
476
(62). We used reverse genetics to generate a clade 1 H5N1 virus harboring the H274Y mutation
477
in NA (derived from A/crested eagle/Belgium/01/2004), while the HA segment was derived from
22
478
NIBRG-14 and the remaining six segments from PR/8, resulting in H5N1 H274Y virus. Next, we
479
determined if N1-3-VHHm, N1-5-VHHm, N1-3-VHHb, N1-5-VHHHb, N1-3-VHH-Fc and N1-5-
480
VHH-Fc can inhibit this virus. As so far all N1-3-VHH formats performed alike N1-5-VHH formats
481
in vitro, we were surprised that only N1-3-VHH (monovalent and both bivalent formats), but not
482
N1-5-VHH in any format, reduced the growth of H5N1 H274Y (Table 2). Compared to NIBRG-
483
14 ma, the H5N1 H274Y IC50 values were three- to seven-fold higher, but still in the low
484
nanomolar range. Even though the competitive SPR analysis suggested that the N1-3-VHHm
485
and N1-5-VHHm epitopes in NA overlap (Fig. 1F), the contact residues necessary for their
486
binding are probably not identical. We conclude that the three evaluated formats of N1-3-VHH
487
can inhibit growth of H5N1 H274Y viruses in vitro, and that both bivalent formats have
488
enhanced in vitro antiviral activity. In addition, the H274Y mutation seems to be sufficient to
489
abolish the in vitro antiviral effect of the N1-5-VHH formats.
490
Next, we tested the antiviral potential of all N1-3-VHH and N1-5-VHH formats against a
491
pandemic H1N1 2009 virus isolate (pH1N1). We used fetuin and MUNANA (AVINA assay) as
492
two alternative substrates for virion-associated NA activity. Using NIBRG-14 ma, and based on
493
MUNANA hydrolysis, monovalent N1-3-VHHm and N1-5-VHHm had significant inhibitory
494
activity, compared with the negative controls SA-VHHm (P < 0.05) and PBS (P < 0.01), although
495
this tendency was not significant in the fetuin assay (Fig. 3A). We attribute these differences
496
between the two assays to the type of substrate used: small molecule MUNANA versus fetuin,
497
which is a large protein carrying multiple sialylated glycans. In the case of fetuin, HA plays an
498
important role in recruiting NA to the substrate, which may alter the accessibility of the N1-VHHs
499
to the NA target. We found that the bivalent molecules N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc
500
and N1-5-VHH-Fc significantly inhibited NA activity for both substrates (P < 0.01, Fig. 3A).
501
In line with our previous results using H5N1 H274Y IAV, only N1-3-VHHb and N1-3-VHH-Fc
502
showed NAI activity using both substrates. (P < 0.05, or P < 0.01) (Fig. 3B). N1-5-VHHb and
23
503
N1-5-VHH-Fc showed inhibitory potential against pH1N1 in both assays. (P < 0.05) (Fig. 3C).
504
We could not detect binding of the N1-VHHs to A/NewCaledonia/20/99 or inhibition of NA
505
activity of this virus (Fig. 3D and data not shown). These AVINA and fetuin results for NIBRG-14
506
ma and H5N1 H274Y NA are in accordance with the N1rec inhibition and the plaque size
507
reduction assays mentioned before (Table 2). In addition, both bivalent N1-5-VHHb formats
508
inhibited pH1N1 virion-associated NA activity, suggesting a degree of intra subtype inhibitory
509
effect of the N1-VHHs.
510
N1-3-VHHm and N1-5-VHHm treatment reduce early morbidity, but not viral lung titer, in
511
H5N1-challenged mice
512
We next evaluated the in vivo antiviral effect of N1-3-VHHm and N1-5-VHHm. The endotoxin
513
levels of the VHH preparations that were used in vivo were low and comparable for the NA-
514
specific VHH proteins and their irrelevant control counterparts (Table 3). In the first experiment
515
we administered intranasally 100 µg of N1-3-VHHm, N1-5-VHHm or N1-7-VHHm to BALB/c
516
mice 4 hours before challenge with 4 LD50 of NIBRG-14 ma. Two groups were included as
517
positive controls (i) a group of mice that received 30 µg of H5-VHHb, a bivalent NIBRG-14 ma
518
HA neutralizing VHH (36), and (ii) a group of mice that received daily oral administration of
519
Oseltamivir at a high dose (45 mg/kg/day). Mice treated with N1-3-VHHm or N1-5-VHHm
520
showed significantly reduced morbidity at 72 and 96 h after infection, compared with the groups
521
treated with N1-7-VHHm and PBS (P < 0.001) (Fig. 4A). Four days after infection, the mice were
522
sacrificed to determine lung virus titers by endpoint dilution in a TCID50 assay. All treated groups
523
of mice (N1-3-VHHm, N1-5-VHHm, N1-7-VHHm, SA-VHHm, PBS and Oseltamivir), except the
524
H5-VHHb group, had a high lung virus load ranging from 104.75 to 106.48 TCID50/ml (data not
525
shown). We also measured the amount of viral RNA in the lung homogenates using a genome
526
strand-specific RT-qPCR method. Except for the samples derived from the H5-VHHb treated
527
mice (P < 0.001), all the N1-VHH treated groups showed high viral RNA levels 96 h after
24
528
infection, comparable to those in the PBS-treated group (Fig. 4B). NA activity in the lung
529
homogenates of the treated groups, measured by AVINA, indicated that there was no difference
530
between the N1-VHH and PBS treated groups, whereas NA activity was strongly reduced in the
531
H5-VHHb and Oseltamivir groups (Fig. 4C). These results are in line with the RT-qPCR results
532
and confirmed the presence of a high viral titer in the lung homogenates of the N1-VHH groups,
533
despite low morbidity. We speculate that the N1-VHH treatment did not have a major impact on
534
virus entry in this in vivo challenge model, but rather reduced virus spread within the lung
535
compartment. A recent systems analysis of influenza A virus-associated pathology in the
536
mouse, revealed a positive correlation between virus spread in the lung tissue and high
537
pathogenicity irrespective of the number of infectious virus particles that were produced (63). In
538
addition, shearing forces that are generated during the preparation of lung homogenates may
539
have released virions from the cell membranes, of which the release was hindered in the intact
540
lung by NA-inhibitory nanobodies. We conclude that intranasal administration of N1-3-VHHm
541
and N1-5-VHHm prevents body weight loss after challenge with NIBRG-14 ma virus during the
542
early stage of H5N1 IAV infection, without apparent decrease in viral titer in the lung
543
homogenates.
544
Protection by bivalent N1-VHHb against H5N1 challenge
545
The in vitro results indicated that the potency of the bivalent formats of the inhibitory N1-VHHs
546
against the tested H5N1 viruses was 30- to 240-fold higher than their monovalent counterparts
547
(Table 2). We therefore assessed if this heightened antiviral effect would also be reflected in an
548
in vivo challenge experiment. We first determined the protective potential during the early
549
stages of H5N1 infection. Four hours before challenge with 4 LD50 of NIBRG-14 ma, groups of
550
BALB/c mice were intranasally given 60 µg of N1-3-VHHb, N1-5-VHHb, or BL-VHHb (a bivalent
551
VHH directed against the irrelevant bacterial target β-lactamase), and 84 µg of N1-3-VHH-Fc,
552
N1-5-VHH-Fc, or GP4-Fc (a plant-produced Coronavirus GP4-Fc fusion protein). Treatment with
25
553
N1-3-VHHb or N1-3-VHH-Fc significantly improved morbidity 72 and 96 h after infection (hpi)
554
compared with the negative control groups GP4-Fc and PBS (P < 0.001) (Fig. 4D). The
555
protection against weight loss was comparable to that observed with the positive controls (H5-
556
VHHb and Oseltamivir, the latter given daily at 45 mg/kg/day by gavage). Treatment with the
557
bivalent formats of N1-5-VHH resulted in reduction of morbidity that was significant at 60, 72
558
and 96 hpi compared with the negative controls (P < 0.001) (Fig. 4D). Determination of the lung
559
virus load on day four after challenge revealed that all challenged groups, except the H5-VHHb-
560
treated mice (no virus detectable), had high and comparable lung virus loads (data not shown).
561
In addition, the NA activity in the lung homogenates of the groups treated with N1-VHH bivalent
562
formats was assessed by an AVINA assay. The lung homogenates treated with bivalent N1-
563
VHHs (N1-3-VHHb, N1-5-VHHb, N1-3-VHH-Fc and N1-5-VHH-Fc) showed 2-5 fold decrease in
564
NA activity when compared to the BL-VHHb (P < 0.001), GP4-Fc (P < 0.05, or P < 0.01) and
565
PBS treated groups (P < 0.01) (Fig. 4E). This could be attributable to residual inhibitory bivalent
566
N1-VHHb in the lung preparation, possibly due to an increased half life of the bivalent N1-VHHs,
567
relative to N1-VHHm, for which no significant reduction in lung associated NA activity was
568
demonstrated (Fig. 4C). Taken together, treatments with bivalent formats of N1-3-VHH and N1-
569
5-VHH improve protection during the first four days following NIBRG-14 ma challenge and
570
reduce the lung associated NA activity, a surrogate for viral load, in H5N1-infected mouse lungs.
571
Dose-dependent protection against H5N1 challenge
572
To test if prophylactic administration of N1-3-VHHb, N1-3-VHH-Fc, N1-5-VHHb or N1-5-VHH-Fc
573
can protect mice against a lethal (4 LD50) challenge with NIBRG-14 ma, we repeated the
574
protection studies and followed the mice for two weeks. We focused on the most potent N1-
575
VHHb bivalent formats and assessed their protective efficacy in a dose-response experiment.
576
Groups of four BALB/c mice were treated intranasally with 60, 12, 2.5 or 0.5 µg of N1-3-VHHb
577
or N1-5-VHHb. In parallel, another group was treated daily by oral administration of Oseltamivir
26
578
(45 mg/kg/day). Negative control groups were treated with 60 µg of BL-VHHb or with PBS
579
before challenge. Mice that had received 60 µg of N1-3-VHHb, N1-5-VHHb or BL-VHHb before
580
the challenge also received a second intranasal dose of 60 µg of the same bivalent VHH on day
581
6 after the challenge. All mice from the PBS and BL-VHHb treatment groups died 9-10 days
582
after infection (Fig. 5C and D). In contrast, treatment with Oseltamivir and intranasal high dose
583
N1-3-VHHb or N1-5-VHHb (60 µg) protected the mice against lethality. In agreement with this,
584
Oseltamivir and N1-5-VHHb (60 µg) reduced post-challenge body weight loss compared to
585
control treated mice (Fig. 5A and B). Although administration of the highest amount of N1-3-
586
VHHb prevented death, it did not affect body weight loss. A single intranasal dose of 12 µg or
587
2.5 µg of N1-3-VHHb or N1-5-VHHb provided partial protection against mortality but failed to
588
reduce morbidity (Fig. 5A-D).
589
We next evaluated protection against a potentially lethal challenge with NIBRG-14 ma by prior
590
single dose intranasal administration of the plant-produced N1-3-VHH-Fc and N1-5-VHH-Fc
591
formats. N1-3-VHH-Fc (80 µg and 17 µg) as well as Oseltamivir prevented death following
592
challenge with NIBRG-14 ma (Fig. 6C), whereas treatment with 3.5 µg of N1-3-VHH-Fc
593
prevented death of some animals. However, challenge was associated with significant body
594
weight loss for all doses and treatments (Fig. 6A and 6B).
595
When comparing the protective efficacy of the two bivalent formats of N1-3-VHH and N1-5-
596
VHH, it appeared that the Fc moiety in the N1-VHH-Fc formats provides an extra protective
597
effect against morbidity (compare 60 µg N1-3-VHHb with 84 µg N1-3-VHH-Fc in figures 5A and
598
6B) as well as against mortality following NIBRG-14 ma challenge (Fig. 5 and 6). We conclude
599
that a single dose of both bivalent N1-3/5-VHH formats can protect mice against a potentially
600
lethal challenge with an H5N1 virus, and that Fc-fused VHHs provide better protection,
601
suggesting that Fc Receptor binding molecules contribute to protection.
27
602
N1-3-VHHb and N1-3-VHH-Fc protect against challenge with Oseltamivir-resistant H5N1
603
Our in vitro analysis demonstrated that monovalent and bivalent formats of N1-3-VHH, but not
604
N1-5-VHH, reduced the growth of Oseltamivir-resistant H5N1 H274Y virus (Table 2). To
605
evaluate these findings in vivo, groups of six BALB/c mice received 30 µg of N1-3-VHHb, N1-5-
606
VHHb or BL-VHHb by intranasal administration 24 h before challenge with 4 LD50 of NIBRG-14
607
ma or 6 LD50 of H5N1 H274Y ma. In parallel, a group was treated by daily oral administration of
608
Oseltamivir (1 mg/kg/day), a dose that has been reported to fully protect laboratory mice against
609
challenge with highly pathogenic H5N1 (64). In line with our previous results, mice treated with
610
N1-3-VHHb, N1-5-VHHb or Oseltamivir suffered from substantial but transient weight loss, but
611
they survived challenge with NIBRG-14 ma virus, whereas mice receiving PBS or BL-VHHb
612
died before day 10 after challenge (Fig. 7). This result demonstrates that a single intranasal
613
administration of N1-3-VHHb or N1-5-VHHb is sufficient to protect against a subsequent lethal
614
challenge (24 h later) with an H5N1 virus. Following challenge with H5N1 H274Y virus, the N1-
615
3-VHHb treated mice were severely sick, but all mice survived (Fig. 7A and 7C). All other
616
groups, including the Oseltamivir treated mice, died after challenge with the H5N1 H274Y virus
617
by day 10 after infection (Fig. 7A and 7C). Taken together, bivalent N1-3-VHHb treatment of
618
mice before challenge with an Oseltamivir-resistant H5N1 virus does not control morbidity
619
adequately but results in full recovery and survival. Furthermore, bivalent N1-5-VHHb does not
620
protect mice against this challenge, suggesting that the N1-VHHb formats (i.e. tandem repeats
621
of VHH separated by a short linker) require the NA inhibitory activity for in vivo protection.
622
Finally, we compared the protective potential of intranasally instilled N1-3-VHH-Fc, N1-5-VHH-
623
Fc and N1-7-VHH-Fc in our lethal challenge model. Following challenge with 4 LD50 of NIBRG-
624
14 ma, all mice in the N1-3-VHH-Fc and Oseltamivir groups lost body weight up until day 7 after
625
challenge (Fig. 7B) and then started to recover, and all mice survived this challenge (Fig. 7D). In
626
contrast, all PBS or GP4-Fc treated mice, except one, became morbid and died by 10 days post
28
627
infection (Fig. 7B and 7D). In the N1-7-VHH-Fc group, only one out of six mice died after
628
challenge. This is very surprising because N1-7-VHHm and N1-7-VHH-Fc have no detectable
629
antiviral activity in vitro. Remarkably, in this experiment mice that had received N1-5-VHH-Fc
630
did not lose weight after challenge with NIBRG-14 ma (Fig. 7B). A challenge with 6 LD50 of
631
H5N1 H274Y virus was lethal to all mice except those that had been pretreated with N1-3-VHH-
632
Fc (all mice survived) or N1-7-VHH-Fc (four out of six mice survived), though all animals lost
633
substantial body weight (Fig. 7B and 7D). We conclude that protection against a potentially
634
lethal challenge with Oseltamivir-resistant H5N1 virus can be obtained by a single dose of N1-3-
635
VHH-Fc or with N1-7-VHH-Fc, although the latter construct is less potent. This indicates that the
636
IgG2a Fc moiety contributes to protection, even in the absence of NA inhibitory activity.
637
N1-5-VHHm escape mutants
638
To identify the amino acid residues involved in the binding of N1-3-VHHm and N1-5-VHHm to
639
H5N1 NA, escape viruses were selected and plaque purified after serial passage of NIBRG-14
640
ma virus in MDCK cells, in the presence of 10 IC50 of N1-3-VHHm or N1-5-VHHm, essentially as
641
described before (35). After nine passages, candidate escape mutants were plaque purified and
642
the NA sequences of four (N1-5-VHHm selection) and three (N1-3-VHHm selection) isolated
643
escape viruses were determined. One of the escape viruses that was selected with N1-5-VHHm
644
contained a mutation in the NA coding region that resulted in a change from isoleucine to
645
threonine at position 437 in the deduced amino acid sequence. This substitution is located on
646
the NA surface, relatively close to the active site (Fig. 8A). A conserved NA epitope that is
647
recognized by HCA-2 under denaturing conditions (65, 66) and localized within the NA catalytic
648
site, and the amino acids necessary for binding a recently reported set of mAbs that recognize
649
H1N1 and H5N1 NA (67) do not overlap with this N1-5-VHHm escape mutations (Fig. 8A).
650
Sequence alignment of a set of representative N1 influenza A viruses, including human H1N1,
651
other H5N1 viruses and an avian H6N1 virus showed that I437 is conserved in these NA
29
652
sequences (Fig. 8B). Binding studies using immobilized virus revealed that the I437T escape
653
virus was no longer bound by N1-3-VHHm, N1-3-VHHb, N1-5-VHHm and N1-5-VHHb (Fig 8C).
654
This result is in accordance with the Biacore data, which showed that N1-3-VHHm and N1-5-
655
VHHm compete for binding to recombinant H5N1 NA (Fig. 1F) and suggests that I437 is part of
656
the VHH epitope in NA or stabilizes the conformation of that epitope.
657
Interestingly, the other three plaque purified N1-5-VHHm escape viruses and all three plaque
658
purified viruses that grew in the presence of N1-3-VHHm contained wild type NA. Possibly,
659
these viruses carry compensatory mutations in other viral proteins such as HA as was described
660
before for influenza A virus passaged in vitro in the presence of small molecule inhibitors of NA
661
(68). Taken together, H5N1 viruses that overcome the inhibition by N1-3-VHHm and N1-5-
662
VHHm can be selected in vitro and one way of escape is through the acquisition of a mutation in
663
NA that results in loss of binding to the VHHs.
664
30
665
DISCUSSION
666
NA Abs usually do not virus neutralizing and are thought to exert their antiviral effects further
667
downstream in the IAV infection process. Still, NA-specific antibodies can significantly reduce
668
viral replication and disease severity (69, 70). Ab titers against N1 or N2 have been associated
669
with high NA enzymatic activity in the vaccine preparation, suggesting that the NA content and
670
activity in IAV vaccines predicts immunogenicity (71). NA of human influenza viruses is subject
671
to antigenic drift at a pace that is comparable to that of HA (72). This suggests that NA is
672
subject to intense immune selection pressure, which is surprising given that antibodies directed
673
against NA do not neutralize the virus in vitro and that during natural infections immune
674
responses against HA are more pronounced than those against NA, presumably because HA
675
outnumbers NA on influenza virions. Biologically, HA and NA cooperate and antigenic variation
676
in NA is in part driven by antigenic escape selection pressure on HA (73). Nevertheless,
677
antibodies directed against NA remain underexplored as potential treatment against influenza.
678
Our N1-VHHs are not the first inhibitory mAbs described against H5N1 NA. Recently, the
679
IgG2bκ mAb 2B9 directed against clade 1 A/Vietnam/1203/04 NA showed interclade and
680
Oseltamivir-resistant mutant NAI in vitro (74). In NAI assays using MUNANA and NA-Star
681
substrates, the 2B9 mAb IC50 ranged from 1 to 8 µg/ml against H5N1 clade 1 and clade 2.1
682
virions, and against an Oseltamivir-resistant double mutant H274Y and N294S. In another
683
recent report on IgG2b mAbs targeting multiple N1 NA, intra-subtype mAbs showed a relatively
684
high H1N1 NAI potency (IC50 of 23.6 to 68.1 ng/ml), which was even lower when
685
A/Vietnam/1203/04 H5N1 NAI potency was assessed (1.41-2.42 µg/ml) (67). We used two
686
inhibitory N1-VHH formats to assess NA inhibitory and antiviral potential. Results from our
687
N1rec inhibition assays showed that N1-3-VHHm and N1-5-VHHm IC50 was 425 nM (6.3 µg/ml)
688
and 374 nM (5.6 µg/ml), respectively, and the introduction of bivalency (N1-VHHb and N1-VHH-
689
Fc) resulted in 2-3 log-fold further increase in their inhibitory potential (5.2-125 ng/ml) (Table 2).
31
690
The above mentioned conventional mAbs, which are bivalent, have an H5N1 NAI activity that is
691
similar to our N1-VHHm. We hypothesize that the much smaller size of VHH molecules
692
compared with conventional antibodies compensates for the lack of avidity that is present in
693
conventional antibodies to explain this.
694
Harmsen et al. reported the first VHH targeting neuraminidase (39). In contrast with our work,
695
where we used recombinant NA protein to immunize an alpaca, these authors obtained VHH by
696
immunizing llamas using whole IAV. This strategy yielded mainly HA and NP binders, and
697
required a cross-selection strategy with homologous NA, followed by further selection with
698
recombinant NA to obtain NA binders. This approach resulted in VHH binding to homologous
699
and non-homologous IAV, but only some of the NA cross-binding VHHs showed NA inhibition
700
activity (fetuin substrate). It is also important to point out that the recombinant NA activities used
701
by Harmsen et al. were 316 to 13.5 times lower than our N1rec specific activity (57
702
µmol/mg/min), while their VHH NA inhibition IC50 values ranged from 124 ng/ml to 2 ng/ml, using
703
homologous NA. The apparently lower IC50 values of the Harmsen VHHs (3 to 200 fold lower
704
than for our N1-VHHm) could therefore be explained by a 1-2 log lower NA specific subtype
705
activity. All together, these results suggest that a similar NAI potency range was obtained with
706
our monovalent N1-VHHm. In vitro, monovalent N1-3-VHHm and N1-5-VHHm had an IC50 that
707
was almost identical to that of Oseltamivir for N1rec and for the two Oseltamivir-sensitive H5N1
708
viruses we assayed (table 2).
709
In the MDCK TMPRSS2-based assay, which allowed us to assess multiple rounds of viral
710
replication in the absence of exogenous trypsin, the antiviral effect of bivalent VHH formats (N1-
711
VHHb and N1-VHH-Fc) was clearly enhanced compared with monovalent N1-VHHm molecules
712
(Table 2). For NIBRG-14 ma, the N1-VHH concentration necessary to reduce the plaque size by
713
50% (plaque assay IC50) for N1-3-VHHb and N1-5-VHHb was 7.6 nM (243 ng/ml) or 14.5 nM
714
(464 ng/ml) respectively, which are 240- and 58-fold lower than their monovalent formats. In
32
715
fact, we noticed a consistent 30-fold or higher inhibitory activity of bivalent N1-3-VHHb and N1-
716
3-VHH-Fc compared to monovalent VHHs in a biochemical assay using N1rec and against the
717
three H5N1 viruses tested in vitro. H5N1 H274Y was an exception in that N1-5-VHH mono or
718
bivalent formats failed to inhibit this Oseltamivir resistant virus (Table 2). These results also
719
suggest that the epitope recognized by N1-3-VHHm and N1-5-VHHm is conserved between the
720
NAs from clade 1 and 2 H5N1 viruses.
721
Intranasal administration of a relatively high dose of N1-3-VHHb or N1-5-VHHb (60 μg; 3.3
722
mg/kg) 24 h before H5N1 challenge protected mice against death, but significant body weight
723
was still lost (Fig. 5). In this challenge model, the N1-VHH-Fc formats protected better than the
724
N1-VHHb formats. A single dose of 17 μg (1.1 mg/kg, N1-3-VHH-Fc) or 3.5 μg (0.23 mg/kg, N1-
725
5-VHH-Fc) resulted in 100% survival of mice, and reduced morbidity compared to control
726
treated animals (Fig. 6 and 7). When comparing the molar ratios of N1-VHH-Fc (N1-3-VHH-Fc
727
and N1-5-VHH-Fc) with N1-VHHb (N1-3-VHHb or N1-5-VHHb), we found that approximately 10
728
to 50 times lower amounts of the N1-VHH-Fc formats were necessary to give 100% survival to
729
H5N1 challenged mice. For comparison, also in a mouse model, conventional mAb 2B9 resulted
730
in only partial (50%) protection against H5N1 A/Vietnam/1194/2004 challenge (74). Even so,
731
this required intravenous administration of 500 μg (33.3 mg/kg) of 2B9 1 h before 10 LD50 of
732
HPAIV H5N1, with daily boosts for 5 days. Furthermore, when we compared the 2B9 treatment
733
with our single-dose treatment with N1-VHHb or N1-VHH-Fc, 10- or 150-fold lower amount of
734
mAb, respectively, was required to rescue 100% of H5N1 challenged mice without any boost.
735
When comparing with a study by Wan et al., the lowest prophylactic single-dose of a
736
conventional IgG2a mAb necessary to fully rescue mice from a 10 LD50 H5N1 challenge was 15
737
mg/kg, whereas 0.23 mg/kg of our N1-5-VHH-Fc protected all mice from death after a 4 LD50
738
challenge
739
A/Vietnam/1194/2004) is almost identical to that used in the Wan report (A/Vietnam/1203/2004).
with
NIBRG-14
ma
(67).
The
target
NA
of
NIBRG-14
(derived
from
33
740
In both reports on NA mAbs discussed above (67, 74), the route of administration (intravenous
741
or intraperitoneal) could have been an important factor and it might help explain the apparent
742
superior protection of our N1-VHH formats, which we administered intranasally, since VHHs
743
withstand nebulization. Moreover, it has been shown that anti-RSV Nanobodies give extended
744
protection against viral infection in vivo when administered via a nebulizer. It may be expected
745
that optimization of the delivery will further help to improve N1-VHH efficacy. N1-VHHm
746
treatments did not significantly reduce the virus titers or viral genomic loads in lung
747
homogenates 4 days after infection (Fig. 4C). The weak, direct antiviral activity of our NA-
748
specific VHHs in the in vivo model is in line with the paradigm that NA Abs or NA-inhibitory
749
drugs do not prevent infection.
750
The observation that N1-5-VHHm, N1-5-VHHb and N1-5-VHH-Fc did not have any antiviral
751
activity against the Oseltamivir-resistant H5N1 H274Y virus in vitro was surprising since in the
752
SPR assay N1-3-VHHm and N1-5-VHHm bound to overlapping epitopes in NA or sterically
753
hindered each other’s binding (Fig. 1F). This discrepancy was also observed in vitro for N1-7-
754
VHHm, whereas N1-7-VHH-Fc did protect mice against challenge with H5N1 H274Y virus (Fig.
755
7). The latter observation indicates that protection by NA-based antibodies does not require the
756
NAI activity of these antibodies, provided that an Fc domain is present to contribute Fc
757
Receptor-dependent effector functions. The finding that also N1-7-VHH-Fc had in vivo antiviral
758
activity despite lack of NAI activity is in line with this (Fig. 7B and 7D). Mouse IgG2a is one of
759
the most potent Ab isotypes to induce antibody-dependent cellular cytotoxicity or complement-
760
dependent cellular cytotoxicity, due to its high affinity for FcγRIII and –IV and especially for
761
FcγRI, which binds exclusively to IgG2a. IgG2a has an intermediate affinity for FcγRIV that is
762
one order of magnitude higher than its affinity for the inhibitor receptor FcγRIIB (75).
763
The VHH CDR3 is a major region responsible for the contact of VHH with antigen (25, 76). All
764
NA inhibitory N1-VHHs reported here (N1-1-VHHm, N1-3-VHHm, N1-5-VHHm and N1-6-VHHm)
34
765
have a predicted electronegatively charged protruding paratope that corresponds to their CDR3
766
(Fig. 1C and 1D). The CDR3 sequences of N1-3-VHHm (HGXXHGHGXHX, 10 residues) and
767
N1-5-VHHm (HXHXHHXHXH, 12 residues), where "H" is a hydrophobic residue and "X" is an
768
aspartic or glutamic acid, might be involved in the direct contact with their epitope or epitopes.
769
When comparing N1-3-VHHm and N1-5-VHHm CDR3 sequences with the most potent NA
770
inhibitory VHHs reported by Harmsen et al., we found similarities in their CDR3 sequences. For
771
example, the CDR3 sequences of one N8-binding VHH (UHXHHHGHGUHHXUPHXH, 18
772
residues) and one N2-binding VHH (HHXHGHHGGHGHUHXH, 16 residues), where “U” is a
773
polar uncharged residue, shared similarities with the N1-3-VHHm and N1-5-VHHm CDR3s.
774
The available crystal structures of mAbs in complex with NA are all for the group 2 NA,
775
specifically N2 and N9 (77-80). These structures showed that these mAb epitopes rarely include
776
conserved amino acid residues from the catalytic site; rather, they target amino acids in loops
777
surrounding the NA catalytic site, also described as the major antigenic regions of NA. It seems
778
that the N1-5-VHHm epitope is partially shared with other N1 NA molecules, as the pandemic
779
H1N1 NA activity inhibition suggests (Fig. 3).
780
Plant-produced Abs have been considered a promising and effective alternative for Ab
781
production by recombinant yeast or mammalian cell culture systems. Our N1-VHH-Fc is the first
782
reported plant-produced Ab against an influenza antigen. Their scalability, robustness and
783
speed of production are major advantages, even though the glycosylation patterns of the plant-
784
produced Abs (rich in high mannose residues) are different from those of the Abs produced in
785
mammalian expression platforms (81). The use of plants for Ab production also presents an
786
interesting approach for developing countries: long storage life, relative ease of purification or
787
delivery, and use of established agricultural techniques are very attractive. To our knowledge,
788
only three single domain Abs produced in plants have been reported as stably transformed lines
789
(82-84), but their yields were very low. Results from our lab demonstrated that the Fc moiety
35
790
significantly improved VHH accumulation in seeds (compared to the monomeric VHH alone)
791
with expression levels of 10-20% of total seed proteins (54, 85).
792
HPAIV H5N1 remains a public health threat. The experimental adaptation of H5N1 virus for
793
airborne transmission in ferrets, with mutations in HA and PB2, warns of the possibility of
794
human-to-human transmission of zoonotic HPAIV (1, 2). Should the HPAIV H5N1 become
795
transmissible among humans, NA targeting could be part of an effective strategy to mitigate
796
disease caused by such a virus. In addition, an interesting prophylactic and therapeutic
797
approach that deserves further validation is the combination of VHH targeting IAV NA, HA or M2
798
proteins in cocktails or biparatopic bivalent VHH constructs that could diminish the antigenic drift
799
in IAV, and lead to much higher selective pressure against IAV VHH.
36
800
Acknowledgements
801
The authors gratefully appreciate the excellent technical support of Tine Ysenbaert, Frederick
802
Vervallle and Jonah Nolf. We thank Dr. Thierry Van den Bergh from the Veterinary and
803
Agrochemical Research Centre in Brussels (Belgium) for providing cDNA of Neuraminidase of
804
A/crested eagle/Belgium/01/2004 (H5N1) influenza virus, Dr. Wolgang Garten from Philipps-
805
Universität Marburg, Germany for MDCK TMPRSS2 cells, Dr. John Wood from the UK National
806
Institute for Biological Standards and Control for providing NIBRG-14 and -23 virus, Dr. Alan
807
Hay (MRC National Institute for Medical Research, Mill Hill, London, UK) for providing A/New
808
Caledonia/20/99 virus, and St. Jude Children's Research Hospital for providing the A/PR/8/34
809
based eight-plasmid system for generating recombinant influenza A viruses. FMC was funded
810
by a pre-doctoral grant from the Biotechnology Institute of Flanders (VIB) and supported by
811
IUAP BELVIR project p7/45.
812
37
813
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1120
44
1121
Figure 1. Characterization of monovalent N1-VHH candidates that bind recombinant H5N1-
1122
derived neuraminidase. A. Soluble N1rec derived from H5N1 A/crested eagle/Belgium/01/2004
1123
expressed in Sf9 cells. A diagram of the n1rec Baculovirus expression cassette (top panel);
1124
promoter, Baculovirus basic protein promoter; ssHA, secretion signal of HA; tGCN4,
1125
tetramerizing GCN4-derived leucine zipper; H5N1 NA, extracellular domain of NA (amino acid
1126
residues 53–449). The lower panel shows NA activity in the culture supernatants of different
1127
amounts of AcNPVN1rec-infected cells or mock-infected cells, using MUNANA as a substrate.
1128
RFU: relative fluorescent units. B. Schematic representation of the expression cassettes of
1129
N1rec-binding VHH domains and derived bivalent formats. Bacteria-produced monovalent N1-
1130
VHHm (n1-vhhm), with the amino-terminal periplasmic signal sequence (pelB), the n1-vhhm
1131
coding information, and a carboxy-terminal hexahistidine tag (his)(top panel). Bacteria-produced
1132
bivalent N1-VHHb with two n1-vhhm moieties fused by a flexible linker (llama IgG2c hinge) and
1133
a hexahistidine tag (his)(middle panel). For expression in transgenic Arabidopsis thaliana seeds,
1134
the N1-VHH-Fc insert was cloned into the seed-specific expression cassette of the PphasGW
1135
vector. LB (left border of T-DNA); 3’ ocs (3’ end of the octopine synthase gene); npt II (neomycin
1136
phosphotransferase II open reading frame); Pnos (nopaline synthase gene promoter); Pphas (β-
1137
phaseolin gene promoter); 5’ utr (5’ UTR of arc5-I gene); SS (signal sequence of the
1138
Arabidopsis thaliana 2S2 seed storage protein gene); n1-vhh-fc (coding sequence of the N1-
1139
VHH gene fused to mouse CH2 and CH3 IgG2a); KDEL (endoplasmic reticulum retention
1140
signal); 3’ arc (3’ flanking regulatory sequences of the arc5-I gene); RB, T-DNA right border
1141
(lower panel). C. CDR3 sequences of 13 N1rec-binding VHH candidates. The VHH CDR3 is
1142
highlighted by a red dotted box. The colored histogram denotes the homology between the
1143
sequences: red, 100%; orange, 60–80%; green, 40–60 %; light blue, 20–40 %; dark blue,
15448
-
>32896
-
>15448
-
>32896
-
>32896
123 -
0.157 (+/0.206) 0.69 (+/0.231) >15448 1.31 (+/0.605) 1.49 (+/0.746)
2708
7.6 (+/- 3.0)
240
52.2 (+/36.8)
543
14.5 (+/- 11.1)
58
>707.5 >32896 89.4 (+/7.8)
c
Fold
4295 (+/2835.4) 3492 (+/2967.6)
-
>32896
-
>32896
-
24.6 (+/31.9) 23.6 (+/3.8) >32896 24.2 (+/3.7)
174 148
-
>32896
-
324
23.08
79
251
28.14
30
>17083
-
27.03
129
>7900
-
>5260
-
>5260
-
>5260
-
>7900 586.1 (+/120.5)
-
>5260 15030 (+/10039)
-
>5260
-
>5260
-
-
>243000
-
73000
-
-
-
b
NIBRG-23
71
177
Mean inhibitory concentration (IC50) from three independent neuraminidase inhibition
assays using 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and 160 ng of N1rec. b
Mean plaque inhibitory concentration (IC50) from two independent plaque assays performed
in duplicate, in which the VHH concentration reduced the plaque size by 50% compared with mock VHH. c
Fold increase inhibitory potency of the N1-VHH bivalent format compared to its monovalent
format. d
Monomeric VHH against seed storage albumin.
e
Bivalent VHH against bacterial beta lactamase.
f
Coronavirus GP4 protein fused to mouse IgG2a Fc
Table 3. Endotoxin levels in the purified VHH preparations VHH N1-3-VHHm N1-5-VHHm N1-7-VHHm SA-VHHm N1-3-VHHb N1-5-VHHb BL-VHHb N1-3-VHH-Fc N1-5-VHH-Fc N1-7-VHH-Fc GP4-Fc PBS
a
LPS content (EU/ml)a 0.094 (+/- 0.007) 0.096 (+/- 0.017) 0.095 (+/- 0.012) 0.094 (+/- 0.008) 0.092 (+/- 0.005) 0.093 (+/- 0.007) 0.107 (+/- 0.024) 0.100 (+/- 0.020) 0.103 (+/- 0.019) 0.076 (+/- 0.011) 0.100 (+/- 0.016) 0.07 (+/- 0.015)
Endotoxin levels in the purified E. coli (VHHm/b) and A. thaliana (VHH-Fc and GP4-Fc)
derived proteins was determined using Limulus amebocyte lysate-based chromogenic test and calculated relative to an endotoxin standard.
