Ethics statement

All animal experiments were carried out in accordance with the Guidelines for Animal Experiments of the National Institute of Infectious Diseases (NIID). Experimental protocols were reviewed and approved by Institutional Animal Care and Use Committee of the Temasek Life Sciences Laboratory, National University of Singapore, Singapore. (IACUC approval number TLL-10-012).

All experiments involving human H7 strains were performed in a biosafety level 3 (BSL-3) containment laboratory in compliance with CDC/NIH and WHO recommendations and were approved by the Agri Veterinary Authority (AVA) of Singapore.

Viruses and cell lines

Production and characterization of Mab

BALB/c mice were immunized twice subcutaneously at intervals of 2 weeks with BEI (binary ethylenimine) inactivated H7N1 (A/Chicken/Malaysia/94) and adjuvant (SEPPIC, France). Mice were boosted with the same viral antigen, 3 days before the fusion of splenocytes with SP2/0 cells [15]. The fused cells were seeded in 96-well plates, and their supernatants were screened by immunofluorescence assays as described below. The hybridomas that produced the Mabs were cloned by limiting dilution at least three times. The positive Mabs were tested for their hemagglutination inhibition activity as described below. Immunoglobulins from selected positive Mabs were isotyped using a commercial isotyping kit (Amersham Bioscience, England) as described in the manufacturer’s protocol. The hybridoma suspension was harvested 3 days postseeding and cell debris pelleted by centrifugation at 400 g for 10 min, followed by collection of the supernatant and storage at -20°C. Mabs were purified with Montage kit Prosep-G (Millipore) for IgG.

Experimental serum samples

Inactivated AI viruses (Table 1) were emulsified in ISA-70 (SEPPIC, France) adjuvant and injected intramuscularly to the groups of three weeks old white leghorn chickens (n = 4). The booster was given twice at two-week intervals. Sera were prepared from the blood collected 10 days after 1st injection and 2nd injection. Antibody responses to the homologous strains were evaluated by HI as described below. Groups of mice (n = 4) were injected intramuscularly with different inactivated H7 AIVs (Table 1) individually emulsified in adjuvant (SEPPIC, France). The injections were repeated twice at two-week intervals. In addition, guinea pigs were immunized with inactivated H7N1 (A/Chicken/Malaysia/94). Blood was collected 14 days after the 2nd immunization.

Hemagglutination inhibition assay

Hemagglutination inhibition (HI) assays were performed as described previously [16]. Briefly, Mabs were serially diluted (2 fold) in V-bottom 96-well plates and mixed with 4 HA units of H7 virus. Plates were incubated for 30 min at room temperature, and 1% chicken RBCs were added to each well. The hemagglutination inhibition endpoint was the highest Mab dilution in which agglutination was not observed.

Isolation and analysis of escape mutants

The epitope recognized by Mab 62 was mapped by characterization of escape mutants as described previously [9]. Briefly, H7N1 parental viruses were incubated with an excess of Mab for 1 h and then inoculated into 11 day old embryonated chicken eggs. The eggs were incubated at 37°C for 48 h. Virus was harvested and used for cloning in limiting dilution in embryonated chicken eggs and the escape mutants were plaque purified. The HA gene mutations were then identified by sequencing and comparison with the sequence of the parental virus.

Microneutralization assay

Neutralization activity of Mab against H7 strains was analyzed by microneutralization assay as previously described [17]. Briefly, Mab was serially two-fold diluted and incubated with 100 TCID50 of different clades of H7 strains for 1 h at room temperature and plated in duplicate onto MDCK cells grown in a 96-well plate. The neutralizing titer was assessed as the highest Mab dilution in which no cytopathic effect was observed by light microscopy.

H7 baculovirus production

The recombinant baculovirus vector was generated as described previously [18]. The full length HA gene was amplified from H7N7 (A/NL/219/03) reassortant virus in a standard PCR reaction. The amplified HA gene was inserted into the shuttle vector pFASTBacHT A (Invitrogen, San Diego, CA, USA) for expression under the white spot syndrome virus (WSSV) immediate early (ie1) promotor. This expression cassette was integrated into the baculovirus genome within DH10BacTM (Invitrogen, USA) through site specific transposition according to the protocol of the Bac-to-Bac system (Invitrogen). SF9II cells were maintained in SF900II serum free medium (Gibco BRL, USA) at 28°C for recombinant baculovirus synthesis. The recombinant bacmid was then transfected into SF9II cells and the supernatant containing recombinant baculovirus displayed H7-HA (Bac-H7) was harvested at 96 h post-infection.

Dual-function-ELISA

96-well, round-bottom microtiter plates (Nunc, Roskilde, Demark) were coated with 0.5 ug/well of capture MAb 98 in 100 ul of carbonate buffer (73 mM sodium bicarbonate and 30 mM sodium carbonate, pH 9.7) overnight at 4°C or 37°C for 2 h. The plates were washed twice with PBST, followed by two washes with PBS after each incubation with antibody or antigen. The antibody-coated plates were blocked by incubation with 100 ul of blocking buffer (PBS containing 5% milk) for 1 h at room temperature. For antigen detection, the blocked plates were then incubated at 37°C for 1 h with 100 ul of virus-containing samples diluted in PBST. For antibody detection, 50 ul of serum samples mixed with 50 ul of H7 surface expressing baculovirus of 8 HAU were added to the blocked plates for 1-hour-incubation at 37°C. Virus binding or antibody blocking was detected by incubation for 1 h at 37°C with 100 ul of horseradish peroxidase-conjugated detection MAb 62 (800 ng) (in-house labeling; Roche). Chromogen development was mediated by the addition of 100 ul of freshly prepared substrate solution (o-phenylenediamine-dihydrochloride; Sigma). The reaction was stopped with sulfuric acid of 0.1 N, and the optical density at 490 nm was recorded. The antigen detection limit was determined by the optical density value that gave a signal-to-noise ratio of 3. For antibody detection, the OD intensity reduction caused by serum antibodies blocking Mab binding was calculated for each sample dilution by using the formula: % inhibition = [(negative reference serum OD-test serum OD)/(negative reference serum OD-positive reference serum OD)]×100%. To determine the cut-off value of antibody detection, specific pathogen-free chicken sera, mice and guinea pigs were obtained from the Animal Health Biotechnology Serum Bank, Temasek Life Sciences Laboratory, Singapore.

Mab 62 and 98 recognize conserved neutralizing epitopes on H7 AIVs

A panel of Mabs against influenza hemagglutinin was screened for efficient recognition of different strains of H7 viruses. Based on the results of the HI assay and virus neutralization (Table 1), Mab 62 and 98 were selected for further studies due to their high HI activity against a wide range of H7 viruses from birds and humans, including strains from the recent H7N9 outbreak in eastern China. Both the Mabs belong to the IgG1 isotype. The virus neutralizing activity of Mab 62 and 98 was further confirmed to be positive against H7 AIVs. Based on this, the amino acids involved in forming the epitope of Mab 62 and 98 were analyzed using selection of neutralization escape mutants. A/chicken/Malaysia/94 H7N1 virus was used as parental virus for the selection. Sequences of the complete HA genes isolated from multiple escape variants were compared with the parental virus. It was found that mutants generated with Mab 62 have a single mutation on amino acid 175 from Lysine to Glutamate. Mab 98 carried mutations either at amino acid 136 (Ser to Gly), or 137 (Gly to Arg). The numbering of amino acid on HA starts from “ATG” and includes the signal peptide.

Development of the dual-function-ELISA

The dual-function-ELISA was operated as shown in Figure 1. H7 antigen can be detected in an AC-ELISA based on H7 specific Mabs. Mab 62 was randomly selected as the detector antibody and Mab 98 was used as the capture antibody due to their equivalent performance in the reversible use in H7 AC-ELISA. Optimal concentrations of MAbs 62 and 98 for detection and capture were determined by two-way titration of MAb concentrations. The combination that gave the highest signal-to-noise ratio was determined to be 0.5 ug/well of capture MAb 98 and 0.9 ug/well of MAb 62 for detection. The tested virus was considered to be positive with H7 antigen in the dual ELISA when the absorbance was three times higher than that of the non-H7 viruses.

Serum antibodies to H7 can be detected by virtue of their ability to block the recognition of the target epitope by a H7 specific Mab in an ELISA assay. To combine this assay to the AC-ELISA, serum samples were incubated with the fixed amount of recombinant baculovirus, which displays H7 on the virus surface, before being loaded to the plate coated with the capture Mab. H7 antibody titers in samples were determined based on the reduction of the detected H7 baculovirus. Different concentrations of H7 baculovirus were tested before confirming the optimal concentration at 8 HAU. Serum panels from normal or H7 immunized chicken and mice were used to determine the cut-off value. First, a panel of normal serum samples from 16 chicken and 20 mice lacking antibodies to H7 was used to determine the baseline of non-specific reduction in Mab 62 binding to H7 antigen in the dual ELISA. Mean reduction of dual ELISA readings was 6.5% for this serum panel, with a standard deviation (SD) of 7.1. Specific blocking activities can be determined with 95% confidence if a “cut-off value” of ≥30% is set for serum samples. The latter was obtained by adding 3 SD to the mean 6.5% blocking (6.5 + 21.3 = 27.8%). In the test, the dilution factor of each serum sample at was recorded when it presented ≥30% signal blocking rate. Additionally, the blocking rate of each sample diluted at 20 times was recorded for comparison.

Specificity and sensitivity of H7 antigen detection by the dual-function-ELISA

The specificity of H7 antigen detection by the dual ELISA was tested with 6 H7 strains from humans and avian species and 13 representative non-H7 subtype influenza virus strains from different regions and years, including pandemic influenza and avian influenza virus strains circulating in humans (Figure 2). Viruses of H7 or HA subtypes not available in our laboratory were rescued by reverse genetics with the six internal genes from A/Puerto Rico/ 8/34. The reactivity and specificity of H7 antigen detection in the dual-ELISA were examined with 100 ul of PBS containing the H7 strains adjusted to an HA titer of 8. Non-H7 viruses with HA titers of ≥16 were used in order to eliminate false-positive results. No cross-reactivity was observed for any of the non-H7 subtype viruses tested.

The analytical sensitivity of H7 antigen detection in the dual ELISA was determined against four different H7 strains which had absorbance readings ranging from 0.7 to 1.3 at 8 HAU (Figure 3). The three selected H7 viruses were diluted serially for the determination of the detection limit based on virus HA titer. With a cut-off value of 0.2, the detection limit was determined to be 100 ul of sample containing 1 HA titer of virus (equal to TCID₅₀ 10^3.2 of H7N7 A/Netherlands/219/03; TCID₅₀ 10^2.12 of H7N1 A/Chicken/Malaysia/94) for viruses that had average and higher-than-average absorbance, while it was 2 HA titers (equal to TCID₅₀ 10^2.354 of H7N6 A/quail/Aichi/4/09) for viruses that had lower-than-average absorbance. The detection limit of HI test for influenza virus was determined at 2 HAU (100 ul) and subtype cross-reactivity were observed.

Specificity of H7 antibody detection by the dual-function-ELISA

The specificity of the H7 detection by the dual ELISA was investigated using a panel of antisera from experimentally immunized chickens, mice and guinea pigs. Animal sera collected 10 days after the 2nd immunization were first diluted to obtain HI titer of 16 to the homologous virus to normalize antibody concentrations prior to use in EB-ELISA. Sera from chicken immunized with H7N1 influenza viruses (Figure 4) presented ≥85% inhibition in Mab 62 binding, while sera from chickens immunized with H1-H6 and H8-H13 showed maximum blocking of 10%, well below the 30% threshold established for samples containing H7 specific antibodies. No inhibition was detected with sera immunized with wild type baculovirus. Positive inhibition was also observed with all mouse sera from individual immunizations with 4 different H7 strains, indicating the assay is specific to detect H7 antibodies. All animal sera from H7 immunization, including chicken, mouse and guinea pigs, showed positive blocking in the dual ELISA, indicating the assay is effective for sera from any species. These results indicate that the antibody detection in the dual ELISA could positively identify serum samples containing antibodies to H7 without any cross reaction to sera from other subtypes.

Sensitivity of H7 antibody detection by the dual-function-ELISA