The LA4 cell line, which was derived from mouse lung adenoma, was maintained in DMEM/F-12 medium (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin (P/S). Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Paque separation (13) of heparinized whole blood obtained from healthy BALB/c mice (6–8 weeks old). To prepare the ADCC target cells, LA4 cells were transfected with an HA expression plasmid that based on the cDNA fragment of influenza virus strain A/Hong Kong/415742/2009(H1N1)pdm09. Specifically, the full-length HA fragment was cloned into a mammalian expression vector pEAK10 plasmid containing a mouse IgG1 Fc gene (CH2 + CH3) (14). The pandemic H1N1 influenza virus strain A/Hong Kong/415742/2009(H1N1)pdm09 was used for in vitro virus infection; while its mouse-adapted version, A/Hong Kong/415742Md/2009 (H1N1)pdm09 was propagated in embryonated hens’ eggs and utilized for in vivo experiment (15). The viruses were stored in −80°C in aliquot and titrated by standard plaque assay. All experiments with live viruses were conducted using biosafety level 2 facilities as described previously (16).

BALB/c female mice, 6–8 weeks old, were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating procedures of the biosafety level 2 animal facilities and were approved by the Animal Ethics Committee in the University of Hong Kong (17).

Vaccinations were carried out to immunize the mice with E1 or E2 or HA epitopes by DNA priming and protein boost. PBS was used as a negative control. The specified vaccination scheme was listed in Table 1. To prepare the DNA plasmids, either of the E1 or E2 fragment (11) was cloned into the mammalian expression vector pEAK10 as described for the HA plasmid construction. The resultant plasmid DNA (100 μg per mice) was used for DNA priming of the mice by electroporation. To prepare protein for vaccination, recombinant HA, E1, and E2 fusion proteins were expressed in FreeStyle 293FT™ transient expression system (Invitrogen) and purified by protein A affinity (GE Healthcare). Subsequently, proteins were dialyzed and concentrated with Vivaspin 20 centrifugal concentrator (GE Healthcare), followed by protein boosting through intramuscular route. Each mouse received 25 μg protein at each protein boosting. Sera were obtained at day 68 postimmununization before virus challenge. Antibody titers raised against E1, E2, and HA in mouse sera were evaluated by ELISA as previously described with some modifications (18). Mouse sera collected from the PBS-treated group were taken as a background control.

Immunized mice (15 mice/group) were inoculated with five 50% lethal dose (LD₅₀) of mouse-adapted pandemic H1N1 influenza virus by intranasal route, i.e., 1,000 PFU/mouse. Animal survival and body weight were monitored daily for 14 days after virus challenge. A body weight loss of 30% was set as the humane endpoint. Three mice per group were randomly selected and euthanized on day 3 and 5 post-challenge, respectively. Mouse lungs were collected from the euthanized mice. Half of the lung tissues were harvested for virus titration by RT-qPCR methods (19), while the other half were immediately fixed in 10% of PBS buffered formaldehyde for histopathological analyses as described previously (20).

Slides were examined in a blinded manner and scored with a semiquantitative system according to the relative degree of inflammation and tissue damage (21–24). Inflammation was scored as follows: 0, no inflammation; 1, perivascular cuff of inflammatory cells; 2, mild inflammation (extending throughout 25% of the lung); 3, moderate inflammation (25–50% of the lung); 4, severe inflammation involving over one half of the lung.

Antibody-dependent cell-mediated cytotoxicity activity, reflected by the rate of cell death, was measured by a flow cytometry-based assay that described previously with some modifications (11). Generally, the PKH-67 dye (Sigma) was utilized to label the target cells, i.e., HA-transfected LA-4 cells; while 7-Aminoactinomycin D (7-AAD; Invitrogen) was used to stain the dead cells that mediated by ADCC activity. Experimentally, PKH-67-labeled target cells and unlabeled effector cells (i.e., PBMCs) were prepared in RPMI 1640 medium (Gibco) containing 10% FBS and 1% P/S with a cell density of 10⁶ and 10⁸ cells/ml, respectively. Subsequently, 50 μl of target cells were dispensed into a round-bottom 96-well plate, followed by addition of 1 μl of mouse serum. Mouse serum concentration in each group was normalized before addition according to their titers determined by ELISA. One hour after incubation, 50 μl of effector cells were added to incubate with the target cells. Three hours later, another 1 μl of 7-AAD was added to each well before performing the flow cytometry. Cell death was determined with a FACSAria III flow cytometer using the BD FACS software (BD Biosciences). Percent cell death was calculated by software analysis of four identifiable cell populations: live effector cells (no dye), dead effector cells (7-AAD positive), live target cells (PKH-67 positive), and dead target cells (PKH-67 and 7-AAD double positive). Assay controls used to define cell populations included target cells alone (background cell death) and target cells with 1 μl Triton X-100 added (maximum cell death). Percent ADCC was calculated as [(percent experimental lysis − percent spontaneous lysis)/(percent maximum lysis − percent spontaneous lysis)] × 100%, in which percent spontaneous lysis refers to the percent lysis of infected cells with effectors but in the absence of serum, while percent maximum lysis refers to the percent lysis of infected cells with effectors in the presence of 1% Triton X-100. Experiments were performed in triplicate and repeated twice for confirmation.

Immunostaining was performed as previously described to visualize perforin expression in mouse lung tissues (25). Rat anti-mouse perforin (abcam Ab16074) and goat anti-rat Alexa 594 were used as primary and secondary antibodies, respectively. Images were acquired with a Carl Zeiss LSM 780 system.

RNA extraction, reverse transcription, and qPCR were performed as previously described (26, 27). In brief, total RNA was extracted from mouse lung with RNeasy Mini Kit (Qiagen) and reverse transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche). Real time PCR was performed using LightCycler^® 96 (Roche) machine according to the manufacturer’s instructions. Relative gene expression was normalized to the corresponding β-actin values. The sequences of the primers for RT-qPCR are listed in Table S1 in Supplementary Material.

Statistical comparison was performed by Student’s t-test using GraphPad Prism 6. Differences were considered statistically significant when P < 0.05.

In our previous study, we mapped two putative ADCC epitopes, E1 and E2, on the HA head region. By depleting E1 and/or E2 from clinical plasma IgG antibodies, the ADCC activity dropped significantly, which suggested that the two epitopes played potential roles in eliciting ADCC (11). In this study, we sought to confirm the function of these putative epitopes in the induction of ADCC activity using a mouse model. Immunization of mice gave raise to IgG titers against the E1, E2, or HA protein, as quantified by ELISA (Figure 1). A prime/boost immunization strategy was adopted, and mice that immunized with PBS or HA were included as a negative or a positive control, respectively (Table 1). Our results indicated that serum samples from mice vaccinated with E1 (Figure 1A) or E2 (Figure 1B) both exhibited strong dilution-dependent antibody responses, reaching a titer of more than 1:5000. Additionally, using HA as the coating antigen for ELISA, we demonstrated that E1 and E2 sera could bind full-length HA at a comparable efficiency (Figure S1 in Supplementary Material). Taken together, our data suggested that the vaccination was successful and the resultant serum samples could be utilized for further investigations.

Next, ADCC activities in serum samples from E1-, E2-, or HA-immunized mice were evaluated by flow cytometry-based ADCC assays. To this end, the HA-transfected LA4 cells were labeled with the cell-membrane marker PKH67 and utilized as target cells for ADCC-specific antibody binding. Subsequently, the vaccinated mouse serum was added to bridge the interaction between target cells and PBMC effector cells (Figure 2). The presence of ADCC-mediating antibody was determined by analyzing the cytotoxicity rate within the cell mixture that contained the target cells, serum, and effector cells, in which the dead target cell population was revealed by the cell death marker, 7AAD (Figure 3). As shown in Figures 3A–G, sera from the E1-vaccinated mice consistently triggered the highest 7AAD + rate among all evaluated groups, suggesting that a higher percentage of cell lysis was induced in the E1 group in comparison to the other groups. The percentage of cytotoxicity was normalized using the formula reported by Srivastava et al., with spontaneous and maximum cell cytotoxicity rate taken into consideration (11). Quantitatively, sera from the E1-vaccinated mice elicited a significantly increased (P < 0.05) ADCC activity in comparison with the PBS-vaccinated group (Figure 3H). Of note, though sera from the E2-Vaccinated mice triggered a subtle increase in ADCC activity, the difference was not statistically significant (Figure 3H). Intriguingly, albeit HA contained the E1 epitope, sera from the HA-vaccinated group did not induce a significantly elevated cytotoxicity response in comparison to that of the PBS control group (Figure 3H).

Since E1 was capable of inducing ADCC activities, we hypothesized that E1-vaccinated mice could potentially be protected by the elicited ADCC activity after influenza virus challenge. To this end, we inoculated the vaccinated mice with pandemic H1N1 influenza virus in a lethal mouse model (Figure 4A). As shown in Figure 4B, mice in the HA-vaccinated group, as a positive control, demonstrated a substantial reduction of viral load on both day 3 and day 5 post-inoculation in comparison to the PBS-vaccinated group. Importantly, we detected an approximately one log decrease of viral load in the mouse lungs of the E1-vaccinated group in comparison to that of the PBS-vaccinated group on day 5 post-inoculation, while no significant difference could be observed between the two groups on day 3 post-virus challenge (Figure 4B). In addition, the viral load in the lungs of the E1-vaccinated mice was notably lower on day 5 when compared with that of day 3, suggesting that the ADCC effect was triggered between day 3 and 5 post-inoculation (Figure 4B).

In parallel, we measured the survival rate and body weight changes of the mice. As shown in Figure 4C, all mice from the HA-vaccinated group survived the course of infection while all mice received PBS-treatment died, indicating that the virus challenge was successful. Unexpectedly, our results demonstrated that the mice in the E1-vaccinated group succumbed to influenza virus infection at a significantly earlier time (P < 0.0001) post-inoculation when compared with that of the PBS-vaccinated control group (Figure 4C). In line with the survival rate, mice from the E1-vaccinated group suffered from an apparently accelerated weight lost starting on day 3 post-inoculation in comparison to mice from the PBS- and E2-vaccinated groups, although the difference did not reach statistical significance (Figure 4D).

Next, we carried out histopathological examinations on the lung sections of the virus-infected mice. Using uninfected mouse lung tissues as a control (Figures 5I,J), our observation showed that on both day 3 and day 5, mice from the HA-vaccinated group (Figures 5G,H) exhibited ameliorated alveolar morphology changes when compared with those from the E1 (Figures 5C,D), E2 (Figures 5E,F), and PBS (Figures 5A,B) groups. Importantly, the lung pathological scores of mice from the HA-vaccinated group on both day 3 and day 5 were significantly lower than those of the PBS-treated mice (Figures 5K,L); while mice from the E1-vaccinated group suffered from a significantly more dramatic interstitial inflammatory infiltration than that of the PBS-treated mice on day 5 (Figure 5L). This result indicated that the detrimental lung damage of E1-vaccinated mice, possibly triggered by ADCC, might account for the reduced viral load in lungs as well as the earlier drop in survival.

To address whether the severe lung damage in the E1-vaccinated group could be attributed to the ADCC effect, we performed immunofluorescence staining to visualize the expression level of perforin among different mouse groups. Perforin is released by activated NK cells and is known as a marker of ADCC activation (28). As quantitated in Figure 6M, the E1-vaccinated mice (Figures 6D–F) demonstrated the highest perforin expression level in the lung sections amongst the other three groups on day 5 post infection (Figures 6A–C,G–L). However, the mRNA expression level of perforin was not significantly different across all evaluated groups (Figure 6N).

Binding of Fc receptor (FcR) on effector cells triggers the secretion of cytotoxic granules as well as antiviral cytokines and chemokines, such as IFN-γ and TNF-α (4). To investigate if elevated expression of proinflammatory cytokines might contribute to the lung damage, we measured the mRNA expression level of representative cytokines including TNF-α (Figure 7A), IL-1β (Figure 7B), and IFN-γ (Figure 7C) in the mouse lungs samples. Our results showed that the gene expression of all three proinflammatory cytokines were significantly enhanced in the E1-vaccinated group when compared with those of the PBS-treated group (Figure 7), which were in line with the perforin protein expression pattern in Figure 6. Together, our data suggested that the E1 epitope from the HA head domain mediated unfavorable ADCC that resulted in a more severe lung damage and exacerbated mouse mortality despite a successful control of the H1N1 influenza viral load.