. 2019 Oct 22;10(5):e01667-19.
Fcγ Receptors Contribute to the Antiviral Properties of Influenza Virus Neuraminidase-Specific Antibodies
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Fcγ Receptors Contribute to the Antiviral Properties of Influenza Virus Neuraminidase-Specific Antibodies
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Influenza virus neuraminidase (NA) has been under intense study recently as a vaccine antigen, yet there remain unanswered questions regarding the immune response directed toward NA. Antibodies (Abs) that can inhibit NA activity have been shown to aid in the control of disease caused by influenza virus infection in humans and animal models, yet how and if interactions between the Fc portion of anti-NA Abs and Fcγ receptors (FcγR) contribute to protection has not yet been extensively studied. Herein, we show that poly- and monoclonal anti-NA IgG antibodies with NA inhibitory activity can control A(H1N1)pdm09 infection in the absence of FcγRs, but FcγR interaction aided in viral clearance from the lungs. In contrast, a mouse-human chimeric anti-NA IgG1 that was incapable of mediating NA inhibition (NI) solely relied on FcγR interaction to protect transgenic mice (with a humanized FcγR compartment) against A(H1N1)pdm09 infection. As such, this study suggests that NA-specific antibodies contribute to protection against influenza A virus infection even in the absence of NI activity and supports protection through multiple effector mechanisms.
IMPORTANCE There is a pressing need for next-generation influenza vaccine strategies that are better able to manage antigenic drift and the cocirculation of multiple drift variants and that consistently improve vaccine effectiveness. Influenza virus NA is a key target antigen as a component of a next-generation vaccine in the influenza field, with evidence for a role in protective immunity in humans. However, mechanisms of protection provided by antibodies directed to NA remain largely unexplored. Herein, we show that antibody Fc interaction with Fcγ receptors (FcγRs) expressed on effector cells contributes to viral control in a murine model of influenza. Importantly, a chimeric mouse-human IgG1 with no direct antiviral activity was demonstrated to solely rely on FcγRs to protect mice from disease. Therefore, antibodies without NA enzymatic inhibitory activity may also play a role in controlling influenza viruses and should be of consideration when designing NA-based vaccines and assessing immunogenicity.
Fcγ receptor; antibodies; influenza; influenza vaccines; neuraminidase.
Copyright © 2019 Job et al.
Characterization of polyclonal anti-rNA immune serum
in vitro and in vivo. (A) Binding of mouse antisera to Bel/09 rNA. Wells of a 96-well flat-bottom ELISA plate were coated with 0.5 μg/ml of Bel/09 rNA overnight in sodium carbonate buffer. Wells were then blocked with 1% BSA in PBS, and heat-inactivated antiserum was subsequently applied in serial dilutions and incubated for 2 h at room temperature in 0.5 mg/ml BSA in PBST. Binding of antisera was detected with either anti-mouse IgG1, IgG2a, or IgG2b conjugated to HRP. Data represents the average from 2 independent experiments ± standard deviation (SD). OD, optical density. (B) Ability of antisera to inhibit virus infectivity. One hundred TCID 50 of Bel/09 was preincubated with increasing dilutions of heat-inactivated antisera directed toward rNA or rHA of Bel/09 or preimmune serum in triplicate and subsequently added to prewashed confluent monolayers of MDCKs. Cells were incubated for 6 days in the presence of trypsin. Virus replication was detected by a hemagglutination assay with turkey red blood cells. Results are expressed as the percentage of wells that scored positive for agglutination. (C) Antisera directed toward Bel/09 rNA can inhibit the viral NA activity. Bel/09 was incubated with increasing dilutions of heat-inactivated anti-Bel/09 NA sera or PBS antisera, and NA activity was determined at 18 h postincubation on fetuin as described in Materials and Methods. Data represent at least 2 independent repeats and show the average ± SD from one experiment performed in duplicate. (D) Anti-Bel/09 NA serum protects mice in a dose-dependent manner. Mice were treated via the i.p. route with 100 μl of heat-inactivated mock serum or increasing amounts of anti-Bel/09 rNA serum and subsequently infected with 1 LD 50 of Bel/09. Mice were monitored for 14 days for weight loss. Mice were sacrificed if they had lost ≥25% of their original body weight. Survival percentages are indicated on the bottom right-hand side of the graph. Text colors match the legend color for each group. Data are representative of 2 repeats and show the average weight loss over time ± the standard error of the mean (SEM [ n = 3]).
Activating FcγRs are not required to control influenza virus in mice treated with polyclonal anti-NA serum. Fifty microliters of heat-inactivated anti-NA sera (circles) or PBS antisera (squares) were passively transferred via the i.p. route into
Fcer1g or Fcgr1/Fcgr3 knockout (KO) mice. BALB/c WT mice were included as controls. One day later, the mice were challenged with 1 LD 50 of Bel/09 and monitored daily, and any mice that had lost ≥25% of their original body weight were euthanized. Weight loss data represent the mean percentages ± SEM of original body weight over time ( n = 8 to 10, pooled from 2 independent experiments). Survival percentages are indicated in the left-hand corner of the graphs where the text color matches colors indicated in the legend for each group (A). In a separate set of experiments, after passive transfer, KO or WT mice were challenged with 0.1 LD 50 of Bel/09 and viral titers assessed in lung homogenates on day 3 (B) or day 5 (C) by TCID 50. The horizontal lines represent the mean (pooled from 2 independent experiments). The dotted line indicates the detection limit of the TCID 50. ns, nonsignificant. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, by one-way ANOVA.
FcγRs aid in viral clearance but do not significantly impact weight loss in mice treated with N1-C4.
Fcer1g or Fcgr1/Fcgr3 KO mice (white symbols) were treated with 1 mg/kg of N1-C4 (circles) or a mouse IgG1 isotype control (squares) via the intranasal route under isoflurane sedation. BALB/c WT mice were included as controls (black symbols). One day later, the mice were challenged with 1 LD 50 of Bel/09 and monitored daily, and any mice that had lost ≥25% of their original body weight were euthanized. Survival percentages are indicated on the left-hand side of the graphs, where the text color matches colors indicated for each group in the legend. Weight loss data represent the mean percentages ± SEM of original body weight over time ( n = 8 to 10 pooled from 2 independent experiments) (A). In a separate set of experiments, after passive transfer, KO or WT mice were challenged with 0.1 LD 50 of Bel/09, and viral titers were assessed in lung homogenates on day 3 (B) or day 6 (C) by TCID 50. The horizontal lines represent the mean. The dotted line indicates the detection limit of the TCID 50. ns, nonsignificant. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, by one-way ANOVA.
(A) HuN1-7D3 and huN1-C4 bind to recombinant NA from Bel/09. Wells of a 96-well ELISA plate were coated overnight with 0.5 μg/ml of recombinant soluble NA from Bel/09 in DPBS plus Ca
2+/Mg 2+. Plates were blocked with 1% BSA in PBS, and the mouse (left) or human chimera version of N1-7D3 or N1-C4 (right) was applied in increasing concentrations in 0.5% BSA in PBST. Binding was detected with either anti-mouse IgG or anti-human IgG conjugated to HRP as appropriate. OD, optical density. (B) HuN1-7D3 does not mediate NA activity against Bel/09, whereas huN1-C4 does. A constant amount of Bel/09 was incubated with 10, 5, 2.5, or 1.25 μg/ml of either huN1-C4, mouse N1-C4, huN1-7D3, or mouse N1-7D3, and NA activity was determined at 18 h postincubation on fetuin by an ELLA. Data are expressed as the percentage of the virus-alone control. NAI, NA inhibitor. (C) N-glycan profiling of mouse and humanized N1-7D3. N-glycans were isolated from mouse or humanized N1-7D3, labeled with APTS, either untreated or treated overnight with fucosidase, and analyzed by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. A labeled dextran ladder (top panel) and N-glycans from bovine RNase B (data not shown) were included as electrophoretic mobility references. The structures of the N-glycans that correspond to the indicated peaks are shown on the right. Blue squares, N-acetylglucosamine residues; green circles, mannose residues; yellow circles, galactose residues; red triangles, fucose residues.
(A) HuN1-7D3 and huN1-C4 can protect against morbidity and mortality in humanized FcγR mice. Humanized FcγR mice were treated with 2.5 mg/kg μg of huN1-7D3, huN1-C4, or an isotype control via the i.n. route under isoflurane sedation. One day later, the mice were infected with 1 LD
50 of Bel/09 and monitored daily over 14 days for weight loss (left panels) and survival (right panels). Weight loss data represents the mean percentage ± SEM of original body weight over time, and survival data are shown as the percentage of survival over time ( n = 6 to 8). Weight loss over time was analyzed by two-way ANOVA examining the main column effects, and survival proportions were assessed using a two-tailed, log-rank (Mantel-Cox) test. *, P < 0.05, and ***, P < 0.001, in comparison to isotype-treated mice. (B) Human chimeric monoclonal antibodies can engage with FcγRIIIa expressed on a Jurkat reporter cell line. Bel/09-infected MDCK cells were used as target cells for a reporter assay where huN1-C4, huN1-7D3, and an isotype control were investigated for their ability to engage the FcγRIIIa expressed on the Jurkat cell line. Antibodies were tested in triplicate in 3-fold dilutions starting from a concentration of 100 μg/ml. Fold induction was calculated from the buffer-alone control (left panel), and curves were used to define EC 50 values in GraphPad Prism (right panel). (C) Coomassie-stained nonreducing SDS-PAGE confirming the correct molecular weight of F(ab′) 2 fragments. HuN1-C4 and huN1-7D3 were treated with IdeS enzyme using the FragIT kit according to the manufacturer’s instructions. Human monoclonal antibodies and F(ab′) 2 fragments were run on a 10% SDS-PAGE alongside a molecular marker (MM) to resolve proteins according to their size. The F(ab′) 2 fragment preparations do not contain intact uncleaved antibody. (D) HuN1-7D3 F(ab′) 2 does not protect against Bel/09 infection in humanized FcγR mice. Humanized mice were treated with equimolar amounts of monoclonal antibodies (2.5 mg/kg) or F(ab′) 2 fragments (1.85 mg/kg) in a total volume of 50 μl via the intranasal route 1 day prior to infection with 2 LD 50s of Bel/09. Mice were monitored for weight loss (left panel) and survival (right panel) for 14 days. Mice that had lost ≥25% of their original body weight were euthanized. Weight loss data show the mean percentage ± SEM of original body weight over time and analyzed by a two-way ANOVA examining main column effects. ***, P < 0.001; ****, P < 0.0001. Survival data are the percentage of survival over 14 days ( n = 6) and were assessed by a two-tailed, log-rank (Mantel-Cox) test. **, P < 0.01 compared to all other groups. Data were pooled from two individual experiments.
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Research Support, Non-U.S. Gov't
Antibodies, Monoclonal / immunology
Antibodies, Monoclonal / pharmacology
Antibodies, Monoclonal / therapeutic use
Antibodies, Viral / immunology
Antibodies, Viral / pharmacology*
Antibodies, Viral / therapeutic use*
Antiviral Agents / immunology
Antiviral Agents / pharmacology*
Antiviral Agents / therapeutic use*
Immunoglobulin Fc Fragments / immunology
Influenza A Virus, H1N1 Subtype / drug effects*
Neuraminidase / immunology*
Orthomyxoviridae Infections / drug therapy*
Orthomyxoviridae Infections / immunology
Immunoglobulin Fc Fragments