Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016–2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.


Introduction
The first severe human cases of influenza A(H7N9) virus infection were reported in the spring of 2003 [1]. Phylogeny told us that these viruses originated from a reassortment among different avian influenza viruses [2]; the hemagglutinin (HA) and neuraminidase (NA) segments were derived from H7N3 viruses and N9 viruses, respectively, and the PB2, PB1, PA, NP, M, and NS segments were derived from H9N2 viruses [3][4][5]. The H7N9 virus has continued to infect humans every influenza season, mainly in China, with the fifth wave occurring in the 2016-17 season [6] and a limited number of human cases being reported in the 2017-18 season. In particular, the Yangtze River Delta and Pearl River Delta regions saw large numbers of H7N9 cases in the 2016-17 season [6]. As of 13 December 2018, a total of 1567 laboratory-confirmed human cases and at least 615 related deaths have been reported

Ethics and Biosafety Statements
The research protocol for the experiments with mice for mAb production was approved by and is in accordance with the policies and procedures of Tauns laboratories, Shizuoka, Japan. All experiments with mice for in vivo protection were performed in accordance with the University of Tokyo's Regulations for Animal Care and Use and were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo.
All experiments with H7N9 viruses were performed in biosafety level 3 (BSL3) laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan.

Viruses
The clinical isolate A/Anhui/1/2013 (Anhui/1; H7N9), which was passaged and propagated in eggs [24], was used for the selection of escape mutants and mouse challenge tests. All wild-type and mutant Anhui/1 viruses and the A/Taiwan/1/2017 (H7N9) virus that were rescued from cloned plasmids [25,26] and propagated in eggs or MDCK cells were used for other experiments. All viruses were titrated in MDCK cells by means of plaque assays.

Hybridomas
The hybridomas used in this study were obtained previously [22]. Mice immunized with inactivated Anhui/1 were used for hybridoma production. The reactivity of secreted mAbs from the hybridoma cell lines was first examined by using an ELISA with purified recombinant HA proteins or purified virions of Anhui/1. The subclass of each clone was determined by using a monoclonal sub-isotyping kit (American Qualex, San Clemente, CA).  /4/2006 (Yamagata-lineage), all of which were purchased from Sino Biological, or purified Anhui/1 (H7N9) virus were reacted with each mAb, followed by enhanced chemi luminescence (ECL) Mouse IgG, horseradish peroxidase (HRP)-Linked Whole Ab (GE healthcare, Tokyo, Japan).

Hemaggulitinin Inhibition Assay
Purified antibody (50 µg/mL) was serially two-fold diluted with PBS prior to being mixed with 8 HA units of Anhui/1. Antibody-virus mixtures were incubated for 60 min at room temperature and then mixed with 0.5% chicken red blood cells. After a 60-min incubation at room temperature, hemagglutination was assessed. The minimum mAb concentration for hemagglutinin inhibition was expressed as the HI value (µg/mL).

Virus Neutralization Assay
Purified antibody (50 µg/mL) in quadruplicate was serially two-fold diluted with MEM containing 0.3% bovine serum albumin (BSA-MEM) prior to being mixed with 100 or 200 TCID 50 (50% tissue culture infectious doses) of the indicated viruses at 37 • C for 30 min. The mixtures were inoculated into MDCK cells and incubated for 1 h at 37 • C. BSA-MEM containing N-tosyl-Lphenylalanine chloromethyl ketone (TPCK)-treated trypsin was added to each well and the cells were incubated for three days at 37 • C. The cytopathic effect (CPE) was examined, and antibody titers required to reduce virus replication by 50% (IC 50 ) were determined by using the Reed and Muench formula.

Evaluation of the In Vivo Protective Efficacy of the mAbs in Mice
Baseline body weights of six-week-old female BALB/c mice (Japan SLC) were measured. Three mice per group were intraperitoneally injected with the indicated antibodies at a concentration of 15 mg/kg [27,28]. One day later, the mice were anesthetized and challenged with 10 mouse lethal dose 50 (MLD 50 ) (50 µL) of Anhui/1. Body weight and survival were monitored daily for 14 days. Mice with body weight loss of more than 25% of their pre-infection values were humanely euthanized.

Generation of Escape Mutant Viruses
10-fold serially diluted Anhui/1 was incubated with each mAb (100 µg/mL) for 30 min at 37 • C. The mixture was inoculated to MDCK cells for 1 h at 37 • C. After removal of the inoculum, infected cells were cultured in BSA-MEM in the presence of trypsin (1 µg/mL) and each mAb (100 µg/mL). At two days after infection, CPE was examined and culture media from CPE-positive wells infected with the highest virus dilution were harvested. The collected culture media were incubated with each mAb (100 µg/mL) for 30 min at 37 • C prior to being subjected to a standard plaque assay. Five plaque-purified viruses per mAb were propagated in MDCK cells. The open reading frame of the HA of the plaque-purified viruses was directly sequenced by Sanger sequencing. Amino acid changes identified in three or more plaque-purified viruses were considered as substitutions that were potentially important for the escape from each mAb.

Phylogenetic Analysis
The phylogenetic tree of the 862 HA nucleotide sequences derived from human H7N9 viruses was constructed by using the neighbor-joining (NJ) method with the Kimura two-parameter method and the bootstrap procedure (n = 100) using the MEGA 7.0.26 software. Sequence data were obtained from the GISAID database on 24 April 2018. The sequencing data set used in this study is available upon request.

Virus Rescue
Plasmid-based reverse genetics for virus generation was performed as previously described [29].  [26] or A/chicken/Huaian/003/2015 (H7N9), and six RNA polymerase I plasmids encoding the other six segments of wild-type or high-yield A/Puerto Rico/8/34 (H1N1) [30] were used. All sequences were synthesized based on the sequences in the GISAID database. Each rescued virus was propagated in MDCK cells and stored as a stock virus. The HA gene of all rescued viruses was sequenced to confirm the absence of unwanted mutations.

Molecular Modeling
The structural model of the H7-HA from A/Shanghai/1/2013 (H7N9) (PDB code, 4LCX) was used to assign the amino acid positions with the PyMOL Molecular Graphics System, version 1.3.
Next, we examined whether these 46 mAbs possess hemagglutinin inhibition (HI) activity and virus neutralization activity against Anhui/1 in vitro. Clones #1 through #7 and #28 through #46 showed no HI and no neutralization potency at a concentration of 50 µg/mL, except for clone 11-21-22 (#3), which possessed weak HI activity and no neutralization activity in vitro ( Table 2). Clones #8 through #27 inhibited virus hemagglutination and virus infection at 0.39-12.5 and 0.62-8.84 µg/mL, respectively ( Table 2). These results together with the cross-reactivity data suggest that clones #1 through #7 recognize the HA stem and clones #8 through #27 target the HA head. Clones #28 through #46 failed to inhibit virus infection in vitro; further analysis is needed to determine whether they play a role in vivo.

In Vivo Protective Efficacy of Cross-Reactive Clones
We examined the in vivo protective efficacy of clones #1 through #7 because some anti-HA stem antibodies protect mice by activating Fc-mediated effector functions without virus neutralization [27,31,32]. Mice were intranasally challenged with 10 MLD 50 of Anhui/1 one day after intraperitoneal injection of each clone at a concentration of 15 mg/kg. A mouse mAb against the NP protein of influenza A virus and a neutralizing mAb against the HA head [clone 3-7-9 (#11)] served as negative and positive controls, respectively. All mice that received clone 11-21-22 (#3), 14-24-5 (#6), or 21-12-10 (#7) died within 5-6 days, as did mice that received the anti-NP mAb (Figure 1). Two of the three mice that received clone 3-5-23 (#2) or 18-18-5 (#4) and one of the three mice that received clone 7-20-10 (#1) or 17-3-11 (#5) survived for two weeks after the challenge infection, although all of the mice transiently lost a considerable amount of body weight. Clone 3-7-9 (#11) protected all mice from lethal infection with Anhui/1 with mild body weight loss. These results show that non-neutralizing mAbs have the potential to partially protect mice from H7N9 virus infection independent of the subclass of the mAbs. 10 (#7) died within 5-6 days, as did mice that received the anti-NP mAb (Figure 1). Two of the three mice that received clone 3-5-23 (#2) or 18-18-5 (#4) and one of the three mice that received clone 7-20-10 (#1) or 17-3-11 (#5) survived for two weeks after the challenge infection, although all of the mice transiently lost a considerable amount of body weight. Clone 3-7-9 (#11) protected all mice from lethal infection with Anhui/1 with mild body weight loss. These results show that non-neutralizing mAbs have the potential to partially protect mice from H7N9 virus infection independent of the subclass of the mAbs.

Figure 1.
In vivo protective efficacy in mice. Three mice per group were intraperitoneally injected with the indicated antibodies at 15 mg/kg. One day later, the mice were intranasally challenged with 10 mouse lethal dose 50 (MLD50) of Anhui/1. Body weight and survival were monitored daily for 14 days. A mouse anti-NP mAb at 15 mg/kg served as a negative control.

Acquisition of Mutant Viruses that Escaped from Neutralizing mAbs
To determine the epitope(s) of the neutralizing mAbs, we attempted to obtain mutant Anhui/1 viruses that escaped from each neutralizing clone. We used the egg-passaged virus for escape mutant selection, because virus diversity should be higher in egg-passaged viruses than viruses generated by reverse genetics, making it easier to obtain escape mutants. Mutant viruses that escaped from clones #8 through #23 possessed the G144E mutation in HA and some of them also possessed the V505A mutation ( Table 3). The A135T mutation in HA was found in a mutant virus that escaped from clones #24 through #27 and some other mutations, including L226Q, which was found in three escape mutants, were also detected ( Table 3). The G144E mutation in the HA of Anhui/1 allowed escape from clones 3-9-18-7 (#13), 8-10-16 (#14), 8-13-19 (#15), 10-19-19 (#16), and 17-3-7 (#19). These results, together with HI data, suggest that G144E plus V505A and A135T plus L226Q are likely involved in evasion from recognition. Figure 1. In vivo protective efficacy in mice. Three mice per group were intraperitoneally injected with the indicated antibodies at 15 mg/kg. One day later, the mice were intranasally challenged with 10 mouse lethal dose 50 (MLD 50 ) of Anhui/1. Body weight and survival were monitored daily for 14 days. A mouse anti-NP mAb at 15 mg/kg served as a negative control.

Acquisition of Mutant Viruses that Escaped from Neutralizing mAbs
To determine the epitope(s) of the neutralizing mAbs, we attempted to obtain mutant Anhui/1 viruses that escaped from each neutralizing clone. We used the egg-passaged virus for escape mutant selection, because virus diversity should be higher in egg-passaged viruses than viruses generated by reverse genetics, making it easier to obtain escape mutants. Mutant viruses that escaped from clones #8 through #23 possessed the G144E mutation in HA and some of them also possessed the V505A mutation ( Table 3). The A135T mutation in HA was found in a mutant virus that escaped from clones #24 through #27 and some other mutations, including L226Q, which was found in three escape mutants, were also detected ( Table 3). The G144E mutation in the HA of Anhui/1 allowed escape from clones 3-9-18-7 (#13), 8-10-16 (#14), 8-13-19 (#15), 10-19-19 (#16), and 17-3-7 (#19). These results, together with HI data, suggest that G144E plus V505A and A135T plus L226Q are likely involved in evasion from recognition.