Comparison of antiviral activity between IgA and IgG specific to influenza virus hemagglutinin: increased potential of IgA for heterosubtypic immunity

Abstract

Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be particularly important for heterosubtypic immunity.

Figure 1. SDS-PAGE analysis of purified S139/1 IgA and IgG antibodies.

Equal amounts (2.5 µg) of purified S139/1 IgA and IgG antibodies were analyzed by SDS-PAGE under reducing conditions (5%–20% gradient gel) (A). Polymeric forms of purified S139/1 IgA and IgG antibodies (5 µg) were analyzed under nonreducing conditions (3%–10% gradient gel) (B).

Figure 2. Comparison of binding activities of S139/1 IgA and IgG antibodies.

Binding activities of S139/1 IgA (continuous lines) and IgG (dashed lines) were tested in ELISA. Disrupted viral particles of Aichi/H3, WSN/H1, Adachi/H2, and Maryland/H13 were used as antigens. Data are mean values of duplicate wells. EC₅₀ values calculated based on the ELISA data are shown in Table 1.

Figure 3. Comparison of neutralizing activities of S139/1 IgA and IgG antibodies.

Appropriately diluted viruses were mixed with S139/1 IgA (continuous lines) or IgG (dashed lines) at the indicated dilutions. Neutralizing activities were evaluated by counting the number of plaques formed on MDCK cells. IC₅₀ values calculated based on the neutralization curves are shown in Table 3.

Figure 4. Viral release from infected cells cultured in the presence of S139/1 IgA or IgG antibodies.

After inoculation with Aichi/H3 (A and B), WSN/H1, Adachi/H2, or Maryland/H13 (C and D), MDCK cells were cultured in the presence of S139/1 IgA or IgG antibodies at the concentrations of 1.0 or 0.1 µg/ml (A and B) and 1.0 µg/ml (C and D). Supernatants were collected 6 and 12 hours after infection, and viral proteins of influenza viruses released into the supernatants were detected by Western blotting (B and D). The relative quantity of the M1 protein was calculated based on the band intensity by using Image Lab version 3.0 (Bio-Rad) (A and C). The intensity of the M1 protein bands detected in the control supernatants collected from infected cells cultured without a MAb (w/o MAb) was set to 100%. Experiments were performed 3 times, and averages and standard deviations are shown (A and C). Statistical significance was analyzed by Student's t-test (**p<0.01, *p<0.05). Asterisks placed directly above the bars indicate significant differences compared to respective controls, and asterisks placed between the bars show significant differences between S139/1 IgA and IgG antibodies.

Figure 5. Viral particles deposited on the surface of infected cells cultured with S139/1 IgA.

MDCK cells were infected with Aichi/H3 and incubated for 8 hours in the presence of S139/1 IgA (A and D), IgG (B and E), or in the absence of antibodies (C and F). TEM images of the cell surface are shown at high (A to C) and low (D to F) magnifications. Scale bars represent 1 µm (A to C) and 2 µm (D to F).