Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Head Region of Pandemic H1N1 Influenza Virus Play Detrimental Roles in H1N1-Infected Mice

Abstract

Engaging the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. However, investigation of the ADCC epitopes on the highly immunogenic influenza hemagglutinin (HA) head region has been rarely reported. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes, designated E1 and E2, on the HA head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. Our data demonstrated that sera from the E1-vaccinated mice could induce high ADCC activities. Importantly, the induction of ADCC response modestly decreased viral load in the lungs of H1N1-infected mice. However, the elevated ADCC significantly increased mouse alveolar damage and mortality than that of the PBS-vaccinated group (P < 0.0001). The phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of E1-vaccinated mice after H1N1 influenza virus challenge. Overall, our data suggested that ADCC elicited by certain domains of HA head region might have a detrimental rather than protective effect during influenza virus infection. Thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of ADCC.

Keywords: H1N1 influenza virus; antibody-dependent cell-mediated cytotoxicity; hemagglutinin; lung damage; mice.

Figure 1

Detection of binding activities in mouse serum samples. ELISA was used to measure antibody titers in serum samples collected from the mice vaccinated with E1 (E1-serum), E2 (E2-serum), HA (HA-serum), or PBS (PBS-serum) on day 68. Binding was tested against either the antigen E1-Fc (A) or E2-Fc (B), respectively. Binding intensities were measured at an absorbance of 450 nm. The experiments were conducted in triplicate. Data shown represents the mean values ± SD (n = 6).

Figure 2

Schematic diagram of the antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC activity was determined by a flow cytometry-based assay using two fluorescent dyes. PKH-67, a membrane-labeling dye, was used to specifically identify the HA-transfected target cells. 7AAD was excluded by viable cells but could penetrate the cell membrane of dead or dying cells and intercalate into double-stranded DNA. Briefly, 50 μl of PKH-67-labeled target cells (10⁶ cells/ml) was dispensed into a round-bottom 96-well plate, followed by addition of E1/E2/HA/PBS sera and effector/PBMC cells. Following a 3-h incubation, 7-AAD was added. Cell death was determined on a FACSAria III flow cytometer using BD FACS Diva software (BD Biosciences). Percent cell death was analyzed by the Flowjo software.

Figure 3

Antibody-dependent cell-mediated cytotoxicity (ADCC) responses are enhanced by the sera of E1-vaccinated mice. ADCC activities in serum samples collected on day 68 were tested by flow cytometry-based assays. (A) Gating of LA4 cells. (B) Gating of PKH67 positive cells. The 7AAD positive cell population for the no serum group (C), PBS-vaccinated group (D), E1-vaccinated group (E), E2-vaccinated group (F), and the HA-vaccinated group (G). (H) ADCC activities of serum samples that collected from each group. The percent of cytotoxicity was derived based on our previously described method (11). *P < 0.05 as compared to the PBS-treated group. The results represent mean values ± SD (n = 5).

Figure 4

E1-vaccinated mice are adversely affected by ADCC. (A) Schematic diagram of the virus challenge study. (B) Three mice from each group were euthanized on day 3 and 5 post-inoculation, and lungs were collected for detection of viral loads by RT-qPCR. Results are presented as bar charts with mean values ± SD. Differences between groups were compared using the t test. *P < 0.05 and **P < 0.01 as compared to the PBS-immunized group. Survival rate (C) and body weight (D) of the mice (9 mice per group) vaccinated with PBS (red), E1 (blue), E2 (green), and HA (purple) were monitored for 14 days. The body weight values are shown as means ± SD for the mice that were alive at each time point (***P < 0.0001).

Figure 5

Lungs of E1-vaccinated mice exhibit more severe histopathological changes upon influenza virus infection. Representative histologic sections of the lung tissues from the mice harvested on day 3 and 5 post-inoculation were stained with H&E. The level of inflammatory infiltrate and thickening of the alveolar septum (as alveolar damage) was detected in samples from mice vaccinated with PBS (A,B), E1 (C,D), E2 (E,F), and HA (G,H). Lung tissues from the uninfected normal mice were included for comparisons (I,J). The black arrows indicate inflammatory cell infiltration. Scale bars represent 20 μm. (K,L) Pathological changes were scored as the criteria indicated in Section “Materials and Methods” (*P < 0.05; **P < 0.01).

Figure 6

E1-vaccinated mice express perforin at a higher level than that of PBS-, E2-, or HA-vaccinated mice upon influenza virus infection. Perforin expression (red) from mouse lung tissues on day 5 post-inoculation was immunolabeled with rat anti-mouse perforin followed by goat anti-rat Alexa 594 (A–L). Nuclei were labeled by DAPI (blue). The white arrows indicate site of perforin expressing. Scale bars represent 20 μm. (M) Quantification of the percentage of perforin protein expression of each group. % = (perforin positive cell/total cells) × 100 (**P < 0.01). (N) Quantitative real time RT-PCR comparing perforin mRNA expression levels from mouse lung tissues on day 5 post-inoculation. Data are shown as fold change compared to the perforin mRNA expression level of the PBS group. The results represent mean values ± SD (n = 5).

Figure 7

Proinflammatory cytokines are further upregulated in the E1-vaccinated mice upon influenza virus infection in comparison with those in the PBS-vaccinated mice. Quantitative real time RT-PCR comparing gene expression levels of TNF-α (A), IL-1β (B), and IFN-γ (C) from mouse lung tissues on day 5 post-virus challenge. Data are shown as fold change compared to the expression level of individual gene in the PBS group (*P < 0.05; **P < 0.01; ***P < 0.001). The results represent mean values ± SD (n = 5).