Potential Role of Nonneutralizing IgA Antibodies in Cross-Protective Immunity against Influenza A Viruses of Multiple Hemagglutinin Subtypes

Abstract

IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs.IMPORTANCE Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.

Keywords: IgA; antibody; broadly cross-reactive; budding; cross-protective immunity; hemagglutinin; influenza A virus; nonneutralizing.

FIG 1

Purification of chimeric MAb 5A5 IgG and IgA. Recombinant IgG and IgA antibodies were purified from the supernatant by affinity chromatography. MAb 5A5 (A) and B12 (B) IgA antibodies were further fractionated by GFC with a Superose 6 10/300 GL column. A chromatogram demonstrating absorbance at 280 nm (shown in milli-absorbance units) revealed two major peaks. Fractions covering the two peaks were subjected to BN-PAGE. Equal amounts (5 μg) of purified IgG, mIgA, and pSIgA from MAb 5A5 (C) and B12 (D) were used for BN-PAGE.

FIG 2

Reactivities of MAb 5A5 IgG, mIgA, and pSIgA to HA antigens from various subtypes. Reactivities of MAb 5A5 IgG, mIgA, and pSIgA to recombinant HAs were measured in ELISA. MAb 5A5 was diluted at 0.1 μg/ml before use. HAs of the following IAV strains were used: A/swine/Hokkaido/2/1981 (H1N1), A/Puerto Rico/8/1934 (H1N1), A/Kadoma/4/2006 (H1N1), A/Narita/1/2009 (H1N1), A/Adachi/2/1957 (H2N2), A/Aichi/2/1968 (H3N2), A/duck/Hokkaido/5/1977 (H3N2), A/duck/Czechoslovakia/1956 (H4N6), A/Hong Kong/483/1997 (H5N1), A/duck/Hong Kong/820/1980 (H5N3), A/shearwater/South Australia/1/1972 (H6N5), A/duck/Hokkaido/301/1978 (H7N2), A/seal/Massachusetts/1/1980 (H7N7), A/turkey/Ontario/6118/1968 (H8N4), A/Hong Kong/1073/1999 (H9N2), A/chicken/Germany/N/1949 (H10N7), A/duck/England/1/1956 (H11N6), A/duck/Alberta/60/1976 (H12N5), A/gull/Maryland/704/1977 (H13N6), A/mallard/Astrakhan/263/1982 (H14N5), A/duck/Australia/341/1983 (H15N8), A/black-headed gull/Sweden/5/1999 (H16N3), and A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10). An influenza B virus strain, B/Lee/1940, was used as a negative control. Columns and error bars indicate the means and standard deviations of triplicate wells, respectively.

FIG 3

(A) Reactivities of MAb 5A5 to cHAs between H1 and H17. H1, H17, and cHAs were assessed as described in Materials and Methods. H1 and H17 HAs were derived from A/Puerto Rico/8/1934 (H1N1) and A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10), respectively. Numbers indicate the positions of amino acids according to H3 numbering. (B) Reactivities of MAb 5A5 IgG, mIgA, and pSIgA to the cHAs were measured using ELISA. Columns and error bars indicate the means and standard deviations of triplicate wells, respectively.

FIG 4

Comparison of avidity to HA antigens among MAb 5A5 antibodies. (A) Reactivities of IgG, mIgA, and pSIgA (0.00001 to 1 μg/ml) to recombinant HAs of PR8, Ad2, HK483, SA1, Eng1, and Alb60 were measured using ELISA. Binding dynamics of MAb 5A5 IgG, mIgA, and pSIgA against the recombinant trimeric PR8 HA (B). Sensorgrams were adjusted (x = 0, y = 0: baseline, y = 100: binding) to allow comparisons between different antibody forms in terms of the dissociation rate.

FIG 5

Neutralization tests of MAb 5A5 antibodies. Serial dilutions of MAb 5A5 IgG, mIgA, pSIgA, and positive-control neutralizing MAbs (0.01 to 100 μg/ml) were mixed with the respective IAV strains, followed by plaque assays as described in Materials and Methods. Means and standard deviations of plaque numbers were calculated from three individual experiments. Relative plaque numbers to each control sample (i.e., cells incubated without MAbs) are shown.

FIG 6

Detection of the viral protein in supernatants and lysates of IAV-infected cells. MDCK cells were infected with IAVs at an MOI 2.0 and incubated with or without 5A5 and B12 MAbs (10 μg/ml). The M1 protein in supernatants (A) and cell lysates (B) was detected in Western blotting and beta-actin was also stained for cell lysate samples. The band intensities relative to each control sample (i.e., cells incubated without MAbs) are shown. Each experiment was performed three times, and averages and standard deviations are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) determined using one-way ANOVA, followed by Tukey’s multiple-comparison tests.

FIG 7

Detection of the viral RNA genome in supernatants of IAV-infected cells. MDCK cells were infected with IAVs at an MOI 2.0 and incubated with or without 5A5 or B12 MAbs (10 μg/ml). The viral RNA genome was detected by real-time RT-PCR. Average copy numbers of the viral genome in the supernatant of IAV-infected cells incubated without any MAb were set to 100%. Each experiment was performed three times, and averages and standard deviations are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) determined using one-way ANOVA, followed by Tukey’s multiple-comparison tests.

FIG 8

Electron microscopy of virus particles on IAV-infected cells. DCK cells were infected with IAVs at an MOI 2.0 and incubated for 8 h with or without MAb 5A5 IgG, mIgA, or pSIgA. Randomly selected fields (10 to 20) were observed, and representative images are shown. Scale bars, 500 nm.

FIG 9

NI activity of MAb 5A5 antibodies. Twofold serial dilutions of MAb 5A5 IgG, mIgA, and pSIgA (6.25 to 100 μg/ml) (A) and 10-fold serial dilutions of positive-control antisera (10² to 10⁶-fold dilution) (B) were mixed with the respective IAV strains, followed by ELLA as described in Materials and Methods. Polyclonal chicken antisera against A/duck/Hokkaido/Vac-1/04 (H5N1), A/Singapore/1/1957 (H2N2), A/mallard/Astrakhan/263/1982 (H14N5), and Eng1 (H11N6) were used as positive controls for N1, N2, N5, and N6. Means and standard deviations of NA activity were calculated from triplicate wells. The NA activity values relative to each control sample (i.e., viruses incubated without MAbs) are shown.

FIG 10

Reduced plaque size in the presence of 5A5 MAbs. MDCK cells were infected with IAVs and incubated with or without 5A5 or B12 MAbs (10 μg/ml). Plaques were stained as described in Materials and Methods (A), and plaque sizes were measured for each well (B). Each box with a horizontal black line represents the interquartile range (IQR) and the median. The marks represent outlying plots located over 1.5 × IQR from the upper quartile. Whiskers extend from the highest and lowest values within a fence. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) determined using one-way ANOVA, followed by Tukey’s multiple-comparison tests.