MAb production and selection
MAbs against H5 HA were generated according to the procedure described previously [9]. Briefly, five 6-week-old female BALB/c mice were immunized with purified recombinant H5 HA protein produced in a mammalian expression system (Immune Technology Corp., New York, NY, USA). Details of the protein, referred to as rH5-mammalian, are provided in Additional file 1: Table S1. The first 10-µg dose of immunogen, emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA), was administered subcutaneously. The booster doses, each containing 10 μg of rH5-mammalian in PBS, were given twice by intraperitoneal injection and once intravenously. Three days after the last immunization, splenocytes were isolated and fused with mouse myeloma of the SP2/0 cell line (ATCC, Rockville, MD, USA). The fused hybrid cells were cultured in RPMI-1640 medium containing FBS, L-glutamine, sodium pyruvate, and antibiotics, with hypoxanthine, aminopterin and thymidine (HAT) as the selecting agents. The hybridomas were subcloned by the limited dilution method. The resulting hybridoma cell lines were grown in RPMI-1640 medium with the same supplements as the selection culture medium except for HAT. The reagents used for fusion and hybridoma culture were purchased from Sigma-Aldrich.
The hybridoma culture supernatants were screened for the presence of IgG antibodies against H5 HA using ELISAs that targeted the various H5 HA antigens. They included proteins from a mammalian expression system (Immune Technology Corp.) and a baculovirus-expression vector system (Oxford Expression Technologies Ltd., Oxford, England, UK). In addition, inactivated, H5-subtype AIVs (x-OvO Ltd., Dunfermline, Scotland, UK), certified by Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe; Legnaro, Padova, Italy), were used. The sequences of the HA antigens employed in the positive hybridoma selection originated from the highly divergent H5-subtype influenza viruses. The selected mAbs, for a total of 7 clones, were purified from the hybridoma culture supernatants using a HiTrap Protein G HP column (GE Healthcare, Uppsala, Sweden).
Analyses of selected mAbs
The finally selected, affinity-purified mAbs, denoted G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, were characterized by isotyping, immunoreactivity studies and peptide mapping, as described previously [9]. Isotyping was performed using a commercial kit: ‘Mouse Monoclonal Antibody Isotyping Reagents’ (ISO-2; Sigma-Aldrich). The reactivity range of the selected mAbs was determined using ELISAs against well-characterized recombinant H5 HA proteins produced in mammalian or insect cells (Immune Technology Corp., Oxford Expression Technologies Ltd., respectively) and inactivated, H5-subtype AIVs (x-OvO Ltd.) certified by IZSVe (Legnaro, Padova, Italy). As a result, the H5 HA antigens varied in form (recombinant proteins or influenza viruses), length (HA fragments or full-length HAs), conformation (properly folded or misfolded), oligomerization state (monomeric or at least partly oligomeric) and glycosylation pattern (mammalian or insect). Conformational H5 HA antigens used in the specificity analyses included the sequences of 12 strains of HP or LP and H5-subtype AIVs. Consistently, they had diverse amino acid sequences, especially within the subtype-determining HA1 subunit, which was confirmed by a homology search against the immunogen (BLAST program, NCBI). To ascertain the cross-reactivity of the obtained mAbs, 21 strains of AIVs (x-OvO Ltd.) certified by IZSVe (Legnaro, Padova, Italy) were used for testing. They represented the H1–H4 and H6–H16 subtypes of influenza viruses. Apart from determination of the specificity range, the mAb examination by ELISAs comprised the binding capability assessment and immunoreactivity profiling. Peptide mapping was performed for the Fab and Fc antibody fragments digested with trypsin using a matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometer (Applied Biosystems, Waltham, MA, USA). On this basis, the profiles of the tryptic peptide maps of individual antibody clones were defined.
The newly generated antibodies against H5 HA were also subjected to the HI test with H5N2 and H5N3 LPAIVs (x-OvO Ltd.) as the reference antigens. Among the H5N1, H5N2, H5N3 and H5N9 AIVs previously employed in ELISAs for antibody specificity, the H5N2 virus had the lowest and the H5N3 virus the highest homology to the HA1 subunit of immunogen used in mAb production (Additional file 1: Table S6). In the test, the reference anti-H5N2, anti-H5N3 and anti-H7N7 LPAIV antisera (x-OvO Ltd.) as well as commercial mAbs against H5 HA (Acris Antibodies GmbH, Herford, Germany; Pierce/Thermo Fisher Scientific, Waltham, MA, USA) served as positive or negative controls (Additional file 2: Tables S1 and S2). The test was performed using erythrocytes of specific pathogen-free (SPF) chickens obtained from the Department of Poultry Diseases, National Veterinary Research Institute (DPD NVRI, Puławy, Poland). Details are provided in Additional file 2.
Serum samples
A panel of 358 chicken serum samples comprised the anti-LPAIV reference antisera, experimental antisera specific for H5 HA and anti-H5 HA negative sera. HA antigens used to obtain the antiserum samples had sequences derived from a total of 26 virus strains.
Reference antisera, certified by IZSVe (Legnaro, Padova, Italy), were purchased from x-OvO Ltd. They were produced in the SPF chickens inoculated with 25 strains of inactivated LPAIVs representing the HA subtypes from H1 to H16. The H5-subtype viruses used for chicken immunization were H5N1, H5N2, H5N3 and H5N9 LPAIVs. In this work, 1, 2 or 3 batches of each anti-AIV antiserum were used. Consistently, the set of reference antisera comprised 31 samples. The presence of subtype-specific antibodies in these samples was confirmed by HI assays with homologous virus strains. The values of HI titers were included in the product certificates.
Experimental antisera were from previously presented efficacy trials for the HA protein of the H5N1 HPAIV produced in Escherichia coli [10]. The vaccine antigen, referred to as rH5-E. coli, was refolded and chromatographically purified from inclusion bodies. Immunization studies were performed in the Rossa 1 line of layer chickens. The chickens were purchased from a commercial breeder on the day of hatching and maintained at an experimental poultry house with wheat straw bedding. Vaccines dedicated for commercial flocks were not administered. Eight groups of 3-week-old layers were vaccinated twice with 25 µg, 15 µg, 10 µg, or 5 µg of rH5-E. coli and aluminum hydroxide (alum) adjuvant. In this work, a total of 115 samples collected 1 and/or 2 weeks after the boost were used.
The anti-H5 HA negative sera were from control groups in the immunization studies with rH5-E. coli, including those reported previously [10]. The studies were performed under semifield conditions, described above, using the commercial layers and broilers—namely, the Rossa 1 and Ross 308 lines, respectively. Samples were collected from the nonimmunized chickens at different time points of the experiments. Thus, the obtained panel of 191 samples negative for H5 HA was completed with 18 sera from SPF layers, the White Leghorn line (DPD NVRI, Puławy, Poland), and 1 batch of normal chicken serum (Abcam, Cambridge, England, UK).
Serum samples and their applications in the development and evaluation of EB-ELISA are described in Table 1. Influenza virus strains used to obtain reference antisera against H5 and non-H5 subtype AIVs are listed in Additional file 1: Tables S2 and S3, respectively, together with other details of serum samples. Supplementary data on the vaccine antigen, rH5-E. coli, and the anti-H5 HA experimental antisera are provided in Additional file 1: Tables S1 and S4, respectively.
Table 1. Sera used for H5 EB-ELISA development and preliminary validation.
Chickens
|
Immunogen
|
Serum description
|
Samples [N]
|
HI titer
|
Origin
|
Anti-H5 HA positive (1): reference antisera against LPAIVs of the H5 subtype
|
Used in evaluation of diagnostic sensitivity 1 (Dse 1)
|
SPF
|
H5N1 AIV, inactivated
|
One batch (#1)
|
1
|
1:512 with H5N1 AIV according to the certificate
|
x-OvO Ltd.1a
|
SPF
|
H5N2 AIV, inactivated
|
Three batches (#1, #2 and #3)
|
3
|
1:256 or 1:512 with H5N2 AIV according to the certificates
|
x-OvO Ltd.1a
|
SPF
|
H5N3 AIV, inactivated
|
Three batches (#1, #2 and #3)
|
3
|
1:512 with H5N3 AIV according to the certificates
|
x-OvO Ltd.1a
|
SPF
|
H5N9 AIV, inactivated
|
Two batches (#1 and #2)
|
2
|
1:512 or 1:256 with H5N9 AIV according to the certificates
|
x-OvO Ltd.1a
|
Positive controls (PC) used for repeatability determination
|
SPF
|
H5N2 AIV, inactivated
|
Batch #3 (weak PC)
|
1
|
1:512 with H5N2 AIV according to the certificate
|
x-OvO Ltd.1a
|
SPF
|
H5N3 AIV, inactivated
|
Batch #1 (strong PC)
|
1
|
1:512 with H5N3 AIV according to the certificate
|
x-OvO Ltd.1a
|
Anti-H5 HA positive (2): experimental antisera against HA from H5N1 HPAIV
|
Used in evaluation of diagnostic sensitivity 2 (Dse 2)
|
Commercial layers, Rossa 1 line
|
rH5-E. coli adjuvanted with alum
|
From 69 chickens at 8, 9, 10 or 11 weeks of age; 1 or 2 sampling time points per chicken
|
115 (115)
|
1:8–1:512 with H5N2 AIV, determined at IBA
|
IBA2a
|
Anti-H5 HA negative (1): sera of various origin
|
Used in determination of cutoff value and diagnostic specificity (Dsp)
|
SPF layers, White Leghorn line
|
None
|
From 10 chickens at 9 or 11 weeks of age; 1 or 2 sampling time points per chicken
|
18
|
negative AI status
|
DPD NVRI3
|
Commercial layers, Rossa 1 line
|
None
|
From 30 chickens at 7, 8, 9, 10 or 11 weeks of age; 3–5 sampling time points per chicken
|
130 (63)
|
< 1:8 with H5N2 AIV, determined at IBA
|
IBA2b
|
Commercial broilers, Ross 308 line
|
None
|
From 42 chickens at 3, 5, 5 ½, 6 or 7 weeks of age; 1–4 sampling time points per chicken
|
61 (33)
|
< 1:8 with H5N2 AIV, determined at IBA
|
IBA2b
|
Negative control (NC) used for repeatability determination
|
Different strains and sex
|
None
|
Normal chicken serum, 1 batch (NC)
|
1
|
< 1:8 with H5N2 AIV, determined at IBA
|
Abcam4
|
Anti-H5 HA negative (2): reference antisera against LPAIVs of the non-H5 subtypes
|
Used in evaluation of analytical specificity (Asp)
|
SPF
|
AIVs: H1–H4, H6–H12 and H14–H16 inactivated
|
One batch of each antiserum
|
20
|
1:128 - 1:2048 with homologous AIVs according to the certificates
|
x-OvO Ltd.1b
|
SPF
|
H13N6 AIV, inactivated
|
Two batches (#1 and #2)
|
2
|
1:128 or 1:1024 with H13N6 AIV according to the certificates
|
x-OvO Ltd.1b
|
1 Certified by Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe; Legnaro, Padova, Italy) and purchased from x-OvO Ltd. (Dunfermline, Scotland, UK). Details of reference antisera against (a) H5 and (b) non-H5 subtype AIVs are provided in Additional file 1: Tables S2 and S3, respectively.
2 Collected in (a) the test vaccine and (b) control chicken groups during immunization studies with H5 HA protein produced in bacteria (rH5-E. coli) at the Institute of Biotechnology and Antibiotics (IBA; Warsaw, Poland). The number of samples described previously with HI titers [10] are provided in brackets. Details of the vaccine antigen and experimental antisera against H5 HA are presented in Additional file 1: Tables S1 and S4, respectively.
3 Obtained from the Department of Poultry Diseases, National Veterinary Research Institute (DPD NVRI; Puławy, Poland).
4 (Cambridge, England, UK), Cat. No. b7477.
Hemagglutinin inhibition (HI) assay
HI activity was determined for sera from commercial chickens immunized and nonimmunized with rH5-E. coli, as indicated in Table 1. The reference viral antigen and antisera used in the HI assay were from x-OvO Ltd. Details are provided in Additional file 1: Table S5. Normal chicken serum (Table 1) was analyzed using reference reagents from the Veterinary Laboratories Agency (New Haw, England, UK).
The HI assay was performed according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [11] following the previously described procedure [10]. Briefly, the sera were analyzed with the heterologous A/turk/Italy/80(H5N2) LPAIV strain at an HI unit (HIU) of 1:8 using SPF chicken erythrocytes (DPD NVRI, Puławy, Poland). Each assay included control erythrocytes, the antiserum against H5N2 LPAIV as a positive control and anti-H7N4 and/or anti-H7N7 LPAIV antisera as negative controls. The HI titer was defined as the reciprocal of the highest dilution of serum that caused an inhibition of hemagglutination activity with 4 hemagglutination units (HAU) of the inactivated antigen. In this study, serum HI titers equal to or greater than 1:8 were considered positive. On this basis, each serum sample was scored as positive or negative for H5 subtype-specific antibodies. The results of the HI assay for 115 antisera collected in the layers immunized with rH5-E. coli and 96 sera from the nonimmunized layers and broilers were adapted from our previous paper [10].
Epitope-blocking ELISA (EB-ELISA)
EB-ELISA was performed using the purified recombinant H5 HA protein (aa 17–530, ΔRRRKKR, 6x His) produced in a baculovirus-expression vector system (BEVS; Oxford Expression Technologies Ltd.). Details of the protein, referred to as rH5-BEVS, are provided in Additional file 1: Table S1. The MediSorp plates (Nunc, Roskilde, Denmark) were coated by overnight incubation at 2–8 °C with 50 μL/well of rH5-BEVS at a concentration of 0.5 μg/mL in PBS. The coated plates were washed three times with 300 μL/well of PBS containing 0.05% Tween 20 (PBST; pH 7.4) and then incubated with 200 μL/well of Protein-Free T20 (PBS) Blocking Buffer (Pierce/Thermo Fisher Scientific) for 1 h at room temperature (23 ± 2 °C). After washing two times with 350 μL/well of PBST, incubation buffer (1% BSA in PBS) was applied to the plates at 50 μL or 100 μL per well. Sera at 50-μL volumes were added to the wells with 50 μL of incubation buffer, resulting in a 2-fold sample dilution. The wells with 100 μL of incubation buffer were left without the serum addition to provide a control of maximum mAb binding to rH5-BEVS (mAb control). Each assay for the test sera, listed in Table 1, was performed in the presence of other control samples. The anti-H5N2 and anti-H5N3 LPAIV antisera were the weak and strong positive controls, respectively (Table 1, Additional file 1: Table S2), while the normal chicken serum (Table 1) was a negative control. All sera were analyzed in duplicate. The plates with test and control samples were incubated for 1 h at 37 °C with shaking at 150 rpm and subsequently washed three times with 300 μL/well of PBST. Next, 50 μL/well of G7-27-18 mAb, diluted to 1 μg/mL in Antibody Stabilizer PBS (CANDOR Bioscience GmbH, Wangen, Germany), was applied to the plates, which were then incubated again for 1 h at 37 °C with shaking at 150 rpm and washed three times with 300 μL/well of PBST afterwards. Antigen-bound mAbs were detected using HRP-labeled, anti-mouse IgG (γ-chain specific) antibodies (Sigma-Aldrich). The plates were incubated with 50 μL/well of anti-mouse antibodies, diluted 1:3,500 in HRP-Protector (CANDOR Bioscience GmbH), for 1 h at 37 °C with shaking at 150 rpm and then washed three times with 300 μL/well of PBST. The reactions were developed with 50 μL/well of TMB (Sigma-Aldrich) at room temperature (25 ± 0.1 °C) in the dark for 15 min and subsequently stopped by adding 50 μL/well of 0.5 M H2SO4.
The optical density (OD) was measured at 450 nm (OD450) using a Synergy 2 multidetection microplate reader (BioTek Instruments Inc., Winooski, VT, USA). For each test and control sample, the mean OD450 value was calculated. The reduction of mAb binding to rH5-BEVS caused by individual specimens was expressed as inhibition percentage calculated using the formula: inhibition percentage = 100 − [100×(OD450 of specimen/OD450 of mAb control)].