|Year : 2016 | Volume
| Issue : 4 | Page : 489-494
Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein
A Malik1, VVA Mallajosyula2, NN Mishra1, AP Arukha1, R Varadarajan2, SK Gupta1
1 Reproductive Cell Biology Laboratory, National Institute of Immunology, New Delhi, India
2 Molecular Biophysics Unit, n Institute of Science, Bengaluru, Karnataka, India
|Date of Submission||23-Dec-2015|
|Date of Acceptance||01-Sep-2016|
|Date of Web Publication||8-Dec-2016|
S K Gupta
Reproductive Cell Biology Laboratory, National Institute of Immunology, New Delhi
Source of Support: None, Conflict of Interest: None
Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (KD) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.
Keywords: Avian influenza, H7N9, monoclonal antibodies
|How to cite this article:|
Malik A, Mallajosyula V, Mishra N N, Arukha A P, Varadarajan R, Gupta S K. Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein. Indian J Med Microbiol 2016;34:489-94
|How to cite this URL:|
Malik A, Mallajosyula V, Mishra N N, Arukha A P, Varadarajan R, Gupta S K. Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein. Indian J Med Microbiol [serial online] 2016 [cited 2017 May 24];34:489-94. Available from: http://www.ijmm.org/text.asp?2016/34/4/489/195366
| ~ Introduction|| |
Influenza is responsible for substantial human morbidity and mortality which are accentuated by periodic epidemics due to the emergence of increasingly virulent strains that escape neutralisation. In the past two decades, increasing number of cases with new re-assorted influenza A viruses such as H5N1, H6N2, H7N3, H7N7, H7N9, H9N2 and H10N7 have been detected in humans. The H5N1 and H7N9 infections among these have been associated with alarmingly high mortality, but sustained human-to-human transmission has not been observed. Within a few months after the first human infection was reported, the H7N9 virus had caused 137 cases of infection, including 45 fatalities. The H7N9 influenza A virus causing human infections in China is a re-assortant of H7N3, H7N9 and H9N2 influenza A viruses that have low pathogenicity in poultry. H7N9 virus-contaminated live poultry markets are the major source of human infections. Since 2013, H7N9 virus outbreaks in humans have occurred in three waves. The third wave viruses prevalent in poultry have descended from second wave viruses and both of them are closely related to the H7N9 strains isolated from infected humans. The dominant H7N9 strains have a dynamic evolutionary process to adapt to local environment. The H7N9 virus is currently endemic to China, but its continuous presence in poultry and re-assortment ability might enable it to infect humans and acquire airborne transmission ability in future.
Control of influenza infections has relied mainly on two options, preventive vaccination or therapeutic antiviral drugs. Influenza viruses in the past have acquired resistance to antiviral drugs. In recent reports, clinical isolates of H7N9 have acquired resistance to oseltamivir and peramivir and partially to zanamivir, commonly used for the treatment of influenza. While the influenza vaccine elicits a robust neutralising antibody response against matched strains, the virus accumulates mutations to escape the host immune pressure. Subsequently, the vaccine efficacy drops significantly against these 'drift' variants or mismatched strains of the virus, necessitating vaccine updates., Therefore, there is an urgent need to develop improved detection systems, more potent antiviral drugs and 'novel' vaccines in preparedness against the serious threat posed by H7N9 virus. Currently available methods for influenza A (H7N9) virus detection include culture, real-time reverse transverse polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA).,, Monoclonal antibodies (MAbs) specifically generated against H7N9 viruses have the potential to be used in detection. Furthermore, human/humanised neutralising MAbs can prospectively be used for passive therapy to combat drug-resistant H7N9 virus infection.
Haemagglutinin (HA) is the major envelope glycoprotein of influenza virus and mediates two critical functions in its life cycle: Receptor binding and host-virus membrane fusion. Sequence homology studies of various subtype HA genes show that there is a clear distinction between the extent of amino acid variations within subtype (ranges from 0% to 9%) and between various subtypes (ranges from 20% to 74%). Keeping this in view, MAbs against recombinant HA (rHA) of A/Anhui/1/2013 were generated with an aim to develop influenza A H7N9 subtype-specific antibodies. The MAbs were characterised against an extensive panel of rHA proteins spanning group 1 and 2 influenza A and B viruses and their binding specificity was established. The MAbs bound H7 HA with a high affinity.
| ~ Materials and Methods|| |
Generation of monoclonal antibodies
Inbred female BALB/cJ mice, 6–8 weeks of age, kept under the conventional containment levels at the Small Experimental Animals Facility, National Institute of Immunology, New Delhi, were used. The studies were conducted as per the guidelines of Institutional Animal Ethics Committee. The mice were immunised intraperitoneally (i.p.) with 25 µg rHA protein (H7N9 A/Anhui/1/2013, Sino Biological, Beijing, PR China) supplemented with 5% Montanide™ PetGel A (Seppic, Paris, France). Primary injection was followed by 2 i.p. boosters at 3-week intervals of 10 µg each of rHA protein supplemented with 5% Montanide™ PetGel A. After 7 weeks, splenocytes from the mouse with the highest antibody titers against rHA (as determined by ELISA) were fused with SP2/O-Ag1.4 mouse myeloma cells in 2:1 ratio using 50% polyethylene glycol (Sigma-Aldrich Inc., St. Louis, USA). The hybrid cells were selected by growing the fused cells in HAT selection media (Sigma-Aldrich Inc., St. Louis, USA). Hybrid cell clones secreting MAbs reacting with rHA of H7N9 were identified by screening culture supernatant in ELISA as described later in this section, and single cell clones were obtained by 2–3 rounds of cloning by limiting dilution method. The hybrid cells of interest were also grown as ascites in BALB/cJ mice primed with Pristane (2, 6, 10, 14-tetramethyl-penta decane; Sigma-Aldrich Inc., St. Louis, USA). The antibodies were purified from ascites using Protein-G Sepharose (GE Healthcare Biosciences AB, Uppsala, Sweden) as per the manufacturer's instructions. Antibody isotyping was carried out using Mouse Immunoglobulins Isotyping ELISA kit (BD Biosciences Pharmingen, San Diego, CA, USA) as per the instructions provided with the kit.
Characterisation of monoclonal antibodies
Enzyme-linked immunosorbent assay
Briefly, 96-well ELISA plates (Corning, New York, USA) were coated with 200 ng/well of mammalian-expressed rHA protein of H1N1 pandemic (A/California/07/2009); seasonal H1N1 (A/New Caledonia/20/1999); H2N2 (A/Canada/720/2005); H3N2 (A/Perth/16/2009); B (B/Brisbane/60/2008); H5N1 (A/turkey/Turkey/1/2005); H7N9 (A/Anhui/1/2013) (all from Sino Biological Inc., Beijing, PR China) or ovalbumin as negative control (200 ng/well) in 0.02 M carbonate-bicarbonate buffer (pH 9.5) for 1 h at 37°C followed by overnight incubation at 4°C. Non-specific sites were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (pH 7.4) for 1 h at 37°C. The plates were washed twice with 0.5% Tween-20 in PBS (PBST). Subsequently, either culture supernatant (100 µl/well) from growing hybrid cells or purified MAbs were added and plates further incubated for 1.5 h at 37°C. After three rounds of washing with PBST, HRP-conjugated goat anti-mouse antibody (Pierce, Rockford, IL, USA) was added at an optimised dilution of 1:10,000 and incubated for 1 h at 37°C. Following three washes with PBST, the enzyme activity was determined using 3,3′, 5, 5′-tetramethylbenzidine (TMB; Thermo Fisher Scientific, Massachusetts, USA) as substrate, and the reaction was stopped by adding 50 µl/well of 5N H2 SO4. The absorbance was read at 450 nm. Values are represented as the mean ± standard error of triplicate readings minus the absorbance obtained with ovalbumin-coated wells. The MAbs showing the mean OD which was lower than three times the mean OD obtained with ovalbumin were considered as non-reactive.
To investigate the reactivity of MAbs in Western blot, H7N9 A/Anhui/1/2013 rHA (1 µg/lane) was resolved in 0.1% SDS–10% PAGE and processed as described previously. Briefly, after protein transfer, nitrocellulose membrane was blocked for 2 h in 5% BSA in tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) followed by two washings in TBS with Tween-20 (0.3% Tween-20; TBST). The blots were further incubated with purified MAbs (10 µg/ml) for 1.5 h on a rocker platform at room temperature. After three washings with TBST, HRP-conjugated goat anti-mouse antibody was added at an optimised dilution of 1:2,000 and incubated for 1 h at 37°C. Following five washes with TBST, the blots were developed with 3,3′-diaminobenzidine tetrahydrochloride hydrate (0.5 mg/ml) supplemented with 0.06% H2O2 (Sigma-Aldrich Inc., St. Louis, USA).
Sequence conservation analysis
All non-identical, full-length H7N9 HA protein sequences (n = 46) reported from humans were obtained from the NCBI-Flu Database for sequence conservation analysis. In CLUSTAL, multiple sequence alignment of all the sequences was performed to generate an alignment file. Subsequently, the sequence conservation was mapped onto a crystal structure of H7 HA (Protein Data Bank ID: 4 LN3) using Chimera.,,
Binding affinity measurements using biolayer interferometry
The binding affinity of MAbs with full-length H7N9 A/Anhui/1/2013 rHA (Sino Biological Inc.) was measured by biolayer interferometry (BLI) using an Octet RED96 instrument (Pall ForteBio, CA, USA). The His6-tagged H7N9 A/Anhui/1/2013 rHA in PBST (PBS with 0.05% Tween 20, pH 7.4) was captured on Ni-NTA biosensors. The binding (nm) of H7 rHA (ligand) with a concentration series of each mAb (analyte in PBST) was independently determined. An unliganded sensor (reference sensor) was used as a control for non-specific analyte binding. The sensor surface was regenerated after each binding experiment with 2M MgCl2. The traces were processed using ForteBio Data Analysis Software (version 8.0, Pall ForteBio, CA, USA) and fit globally using a simple 1:1 Langmuir interaction model.
| ~ Results and Discussion|| |
As per the protocol described in Materials and Methods section, we report the generation of MAbs against avian influenza H7N9 A/Anhui/1/2013 rHA. The use of purified HA instead of the whole virus for immunisation allowed us to generate targeted antibodies against H7N9 rHA. Splenocytes from seropositive mice against H7 HA were used to generate hybrid cell clones. Nineteen positive clones were selected after primary ELISA screening. Subsequently, cloning by limiting dilution led to the generation of five stable hybrid cell clones-secreting antibodies against H7N9 rHA. The reactivity of these five MAbs at varying concentrations with H7N9 A/Anhui/1/2013 rHA in ELISA is shown in [Figure 1]. All MAbs showed concentration-dependent reactivity with rHA. MAbs, MA-20, -24, -26 and -36 showed a significant reactivity (>3-fold OD as compared to the mean OD with ovalbumin used as negative control) with rHA even when used at 39 ng/ml [Figure 1]. The antibody isotype and ELISA reactivity of these MAbs with various rHA are shown in [Table 1]. Out of the five MAbs, three were of the IgG1/k isotype (MA-20, -26 and -36), one monomeric IgA (MA-34) and one IgG2b (MA-24). Further, to characterise the binding specificity of the MAbs, their reactivity against a comprehensive panel of rHA proteins from influenza A (both groups 1 and 2) and B viruses was examined. All the antibodies showed strong binding with H7N9 A/Anhui/1/2013 rHA. We detected no cross-group reactivity for any of the reported MAbs except MA-24. Only MA-24 exhibited weak binding with rHAs from heterosubtypic influenza A Group 1 viruses. The binding was evaluated against both pandemic and seasonal H1N1 rHAs. Very weak binding to H2N2 rHA and moderate binding to H3N2 rHA were also detected for only MA-24. Moderate binding of MA-24 to H3N2 might be because of evolutionary relatedness of H3 and H7 HA protein as both belong to influenza A Group 2 viruses. None of the antibodies reacted with rHA from H5N1 A/turkey/Turkey/1/2005 and a representative influenza B virus strain (Victoria lineage). All the five antibodies were also able to detect H7N9 rHA protein (1 µg) in Western blot [Figure 2].
|Figure 1: Reactivity in enzyme-linked immunosorbent assay of monoclonal antibodies generated against H7N9 A/Anhui/1/2013 recombinant haemagglutinin. Enzyme-linked immunosorbent assay plates were coated with recombinant haemagglutinin protein (200 ng/well) corresponding to H7N9 (A/Anhui/1/2013) and ovalbumin (200 ng/ml) as negative control, and varying concentrations of protein-G purified monoclonal antibodies (MA-20, -24, -26, -34, -36) were used to determine their reactivity as described in Materials and Methods section. The Y-axis represents mean ± standard error of the absorbance at 450 nm observed with the respective antibody tested in triplicate at a given concentration minus the absorbance obtained with ovalbumin|
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|Table 1: Enzyme-linked immunosorbent assay reactivity and antibody isotype profile of monoclonal antibodies generated against H7N9 A/Anhui/1/2013 recombinant haemagglutinin|
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|Figure 2: Reactivity profile of monoclonal antibodies generated against H7N9 A/Anhi/1/2013 recombinant haemagglutinin in Western blots. The H7N9 recombinant haemagglutinin was resolved in 0.1% SDS-10% PAGE followed by processing for its reactivity with various monoclonal antibodies in Western blot as described in Materials and Methods section. Panel (a) shows Coomassie-stained SDS-PAGE profile of H7N9 recombinant haemagglutinin (2 μg/lane). Panel (b) shows immunoblot reactivity profile of H7N9 recombinant haemagglutinin (1 μg/lane) developed with respective monoclonal antibodies used at 10 μg/ml. The subpanels i, ii, iii, iv and v represent the reactivity profile with monoclonal antibodies MA-20, 24, 26, 34 and 36, respectively|
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MAbs offer multiple unique advantages, such as high specificity, consistent properties and easy bulk production, which are ideal for research and detection applications. Strong concordance between ELISA reactivity and detection in Western blots proves the high specificity and sensitivity of these MAbs. Thus, they can be easily used for detection purposes in various other systems and can aid in determining subtype specificity with sensitivity. We further analysed the sequence conservation within full-length HA of all H7N9 viruses isolated from humans till date to ascertain the applicability of the reported MAbs. The overall sequence variation in H7 HA is <3% [Figure 3]. Given the high sequence conservation, we speculate that the reported MAbs can be broadly utilised to detect H7N9 infections in humans. Furthermore, elucidating their epitope (s) will aid in understanding their specificity.
|Figure 3: Sequence conservation within H7 HA. The residue conservation among all non-identical, full-length H7N9 human isolates was mapped onto a monomer of H7 HA trimer (Protein Data Bank ID: 4 LN3); (a) HA1 and (b) HA2 subunits were coloured according to the sequence conservation key. The HA1/HA2 subunit of the same monomer is in yellow. As observed, H7 HA sequences reported till date are well conserved, hence represented in blue according to the sequence conservation key. Rest of HA (excluding HA1/HA2) is shown in grey. The figure was rendered using Chimera|
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It is essential to distinguish the virus subtype, as H5 or H7 avian influenza has already acquired varied resistance to currently used antiviral drugs. While H5N1 has acquired resistance to amantadine and rimantadine, H7N9 has additionally also acquired resistance to oseltamivir and peramivir and partially to zanamivir., In future, if any of the avian influenza viruses gain the ability of airborne transmission, quick detection of their subtype and knowing their antiviral resistance will prove advantageous during treatment. The specificity of such detection assay systems comes from antigenic determinants in the virus envelope glycoproteins, HA and NA. Influenza A viruses are classified into 18 HA (H1-H18) and 11 NA (N1-N11) subtypes. We have recently described the generation of highly specific MAbs against H5N1 HA. The MAbs described in this study, having high affinity and specificity to H7N9 HA, may help in developing diagnostic tools that distinguishes between H5N1 and H7N9 infections.
The kinetic parameters for the binding of the MAbs to H7 A/Anhui/1/2013 rHA was further determined by BLI. All the MAbs bound the homologous H7 HA with very high affinity (0.14–25.2 nM) [Figure 4] and [Table 2]. A recent study demonstrated the in vivo efficacy of a neutralising human MAb (VIS410) against H7N9 (A/Shanghai/2/2013) virus challenge in mice. VIS410 bound HA with nanomolar (nM) affinity as determined by surface plasmon resonance. The antibodies reported herein also bind H7 HA with comparable nanomolar (nM) affinity. The promising binding characteristics of the reported MAbs prompt us to screen for in vitro neutralisation and test their in vivo efficacy as monotherapy or in combination with antiviral drug(s) in the future.
|Figure 4: Binding affinity of anti-H7 HA monoclonal antibodies with full-length H7N9 A/Anhui/1/2013 recombinant haemagglutinin determined by biolayer interferometry using an Octet RED96 instrument. The His6-tagged ligand (H7 A/Anhui/1/2013 recombinant haemagglutinin) in Tween-20 in phosphate-buffered saline was captured on Ni-NTA biosensors. A concentration series (trace 1-4: 200, 100, 50 and 25 nM) of the monoclonal antibodies was used to determine the binding affinity with the captured ligand; (a) MA-20 (b) MA-24 (c) MA-26 (d) MA-34 and (e) MA-36. The kinetic parameters were obtained by fitting the data to a 1:1 Langmuir model. The data points are represented by solid circles, and the fits are shown by dashed lines|
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|Table 2: Kinetic parameters for the binding of monoclonal antibodies with full-length H7N9 A/Anhui/1/2013 recombinant haemagglutinin as determined by biolayer interferometry|
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This work was funded by the Tata Innovation Fellowship awarded to SKG by the Department of Biotechnology, Government of India. Financial assistance from National Institute of Immunology is also acknowledged. The funding agencies had no role in study design, collection, analysis or interpretation of the data.
Financial support and sponsorship
This work was funded by the Tata Innovation Fellowship awarded to SKG by the Department of Biotechnology, Government of India. Financial assistance from National Institute of Immunology is also acknowledged.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]