Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Abstract

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.

Keywords: high-speed atomic force microscopy; influenza virus; intranasal inactivated influenza vaccine; secretory IgA.

Fig. S1.

Schematic representation of the preparation and analysis of nasal wash samples from intranasally vaccinated healthy volunteers. (A) Nasal wash samples were concentrated using centrifugal concentrators. The proportions of IgM, IgA, and IgG antibodies to total antibody after concentration were ∼1.5 (±0.2)%, 73.6 (±3.6)%, and 24.9 (±3.6)%, respectively (mean ± SEM). The concentrated nasal wash samples were used as an initial source in various assays to analyze the properties of nasal IgA antibodies. Specifically, the concentrated nasal wash samples were used to examine the molecular size distribution of each antibody and to determine the neutralizing potency and the binding potency to HA molecules of each fraction after separation through GFC. In addition, the IgA antibody fractions, purified from the pooled nasal wash samples by affinity column chromatography and then separated by GFC, were analyzed using several biochemical methods, and the structure and dynamics of IgA molecules in physiological solutions were evaluated by high-speed AFM. The neutralizing potency and the binding potency to HA molecules of each fraction after separation by GFC were analyzed in relation to the differences in molecular size. (B) SDS/PAGE analysis of purified nasal IgA, serum IgA, colostrum IgA, serum IgG, and serum IgM under reducing conditions. Purified nasal IgA was composed of an α-heavy chain (HCα), light chain (LC), SC, and JC. (C) Blue native (BN)/PAGE analysis of purified nasal IgA, serum IgA, colostrum IgA, serum IgG, and serum IgM. Purified nasal IgA was not homogeneous and included at least two distinct oligomeric structures, with approximate molecular masses of ≥500 kDa and >720 kDa.

Fig. 1.

Size distribution of Igs in the nasal wash samples separated by GFC and estimation of the neutralizing potency of each fraction. (A) Fractionation pattern of nasal wash samples on Superose 6 GFC. The amounts of IgM, IgG, and IgA (wt/wt) in each fraction were estimated using ELISA. Values plotted are means, and error bars represent SEM. Vo, void volume. (B) Proportions of the amounts of IgM, IgG, and IgA (wt/wt) to the total amounts of antibodies in each fraction (IgM + IgG + IgA, weight) were estimated. Values plotted are means, and error bars represent SEM. (C) Neutralization titer against the vaccine strain Victoria virus was determined in each fraction separated by GFC of nasal wash samples collected from five participants (a, b, c, d, and e aged 33, 23, 27, 24, and 34 years, respectively, at the time of sample collection). (D–F) Neutralization titer of each fraction against NY virus, Sydney virus, and Brisbane virus also was determined for the same nasal samples collected from the five participants (a–e).

Fig. S2.

Nasal wash samples from six volunteers vaccinated intranasally with an inactivated whole-virion H5N1 virus vaccine were separated by GFC, and neutralizing potency was estimated for each fraction. (A) Schematic presentation of preparation and analysis of nasal wash samples from intranasally vaccinated healthy volunteers. (B) Fractionation pattern of nasal wash samples on Superose 6 GFC. The amount of IgM, IgG, and IgA (wt/wt) in each fraction was estimated using ELISA. Values plotted are means, and error bars represent SEM. Vo, void volume. (C) Proportion of the amount of IgM, IgG, and IgA (wt/wt) to the total amount of antibodies in each fraction (IgM + IgG + IgA, weight) was estimated. Values plotted are means, and error bars represent SEM. (D) Neutralization titer against the vaccine strain A/Indonesia/5/2005 (H5N1) virus was determined in each fraction separated by GFC of nasal wash samples collected from six participants (a–f). (E and F) Neutralization titer of each fraction against A/Viet Nam/1194/2004 (H5N1) virus and A/Laos/JP127/2007 (H5N1) virus also was determined with the same nasal samples collected from six participants (a–f). The profiles against A/Indonesia/5/2005 (H5N1) virus showed higher neutralization titers in polymeric IgA fractions and lower neutralization titers in IgG fractions. Furthermore, the profiles against A/Viet Nam/1194/2004 (H5N1) virus and A/Laos/JP127/2007 (H5N1) virus for nasal wash samples from all of the volunteers showed no neutralization titers in IgG fractions despite detection of neutralization titers in polymeric IgA fractions. These observations suggested that greater cross-reactivity of polymeric IgA to H5N1 viruses than of IgG to H5N1 viruses and polymeric IgA appeared to play a pivotal role in protecting the human nasal mucosa from infection of H5N1 viruses.

Fig. 2.

Determination of binding activity of antibodies to HA molecules in each GFC fraction by ELISA. The IgA ELISA titer (A), IgG ELISA titer (B), and IgM ELISA titer (C) against HA molecules of Victoria virus (H3N2) were determined in each fraction separated by GFC of nasal wash samples collected from five participants (a, b, c, d, and e aged 33, 23, 27, 24, and 34 years, respectively, at the time of sample collection). HA-specific IgA titers were detected in a broad range of fractions and peaked at fractions 21–30, which corresponded to the first of two peaks of neutralization activity. HA-specific IgG titers peaked at fractions 31–37, which corresponded to the second of two peaks of neutralization activity. The IgA ELISA titer (D), IgG ELISA titer (E), and IgM ELISA titer (F) against HA molecules of NY virus (H3N2) were determined in each fraction separated by GFC of nasal wash samples collected from five participants (a–e). The IgA ELISA titer (G), IgG ELISA titer (H), and IgM ELISA titer (I) against HA molecules of Sydney virus (H3N2) were determined in each fraction separated by GFC of nasal wash samples collected from five participants (a–e). Reduction of the peak titers of HA-specific IgA and IgG antibodies against NY HA (which has 96.6% amino acid similarity to Victoria HA) or Sydney HA (which has 92.9% amino acid similarity to Victoria HA) correlated with decreased sequence similarity to Victoria HA.

Fig. 3.

Biochemical characterization of the quaternary structures of nasal IgA. (A) Purified nasal IgA was subjected to GFC separation according to quaternary structures. (B) Blue native (BN)/PAGE analysis of each fraction of gel-filtered nasal IgA. Nasal IgA samples were composed of at least five distinct quaternary structures (arrowheads). (C) SDS/PAGE analysis of each fraction of gel-filtered nasal IgA under reducing conditions. (D) Western blotting analysis of each fraction of gel-filtered nasal IgA using anti-SC, anti-human IgA α-heavy chain (HCα), anti-human IgA1 subclass-specific (HCα1), anti-human IgA2 subclass-specific (HCα2), anti-human light chain λ (LCλ), anti-human light chain κ (LCκ), and anti-JC antibodies. IB, immunoblot. High-mass MALDI-TOF analysis of serum IgA (E), fraction 28 of nasal IgA (F), and fractions 24–26 of nasal IgA (G). (E) With serum IgA, one major peak (arrow) was detected. (F) With fraction 28 (Frac.) of nasal IgA, one major peak (arrow) was detected. (G) With mixtures of fractions 24, 25, and 26 of nasal IgA antibodies, three major peaks (arrows) were detected. a.u., arbitrary unit; Frac., fraction.

Fig. 4.

AFM revealed the quaternary molecular structures of nasal IgA. AFM images of serum IgA (A), fractions 27–28 of nasal IgA (B), and fractions 24–25 of nasal IgA (C) antibodies at low resolution. (Scale bars, 100 nm.) AFM images of serum IgA (D), fractions 27–28 of nasal IgA (E), and fractions 24–25 of nasal IgA (F) at the single particle level. (Scale bars, 20 nm.) Schematic diagrams next to or under the AFM images show presumed structures of monomeric, dimeric, trimeric, and tetrameric IgA. (G) Successive AFM images of the interaction between nasal trimeric/tetrameric IgA and HA ectodomains of influenza virus (arrows). The time stamp is given as min:sec. The frame rate is 0.5 frames per second. (Scale bars, 20 nm.) (Also Movie S3.) Tetra, tetramer; Tri, trimer.

Fig. S3.

AFM observation of dynamics of nasal tetrameric IgA. (A and B) Time-lapse AFM imaging of nasal tetrameric IgA reveals that the radial arms of the four-leaf clover–shaped complex were rocked asynchronously and bent out of the plane defined by the central portion of the complex. The frame rate in A is 0.25 frame per second, and the frame rate in B is one frame per second. (Scale bars, 20 nm.) The time stamp is given as min:sec. (Also Movies S1 and S2.) Frac., fraction.

Fig. S4.

Time-lapse AFM imaging of the interaction between an HA molecule and nasal polymeric IgA. (A) Ectodomains of H1 HA and H5 HA were prepared by digestion of the purified virions with bromelain in the absence of reducing agents; the digest products were analyzed by SDS/PAGE under nonreducing conditions. (B) AFM images of bromelain-released soluble ectodomain of H1 HA at low resolution (Upper Left) and high resolution (Lower Left). (Scale bars: Upper Left, 50 nm; Lower Left, 20 nm.) The bar graph (Right) indicates the height (Z section) along the white line (Lower Left). The height of each HA molecule was about 8 nm, consistent with data from the X-ray crystal structure. (C) Successive AFM images of the interaction between nasal trimeric/tetrameric IgA and soluble ectodomain of H5 HA. The round-edged HA ectodomain (arrow) was trapped by the radial regions of the nasal IgA and moved along with the edge of the IgA. The frame rate is 0.2 frames per second. The time stamp is given as min:sec. (Scale bars, 50 nm.) Tetra, tetramer; Tri, trimer.

Fig. 5.

Comparison of neutralizing potency between Igs with different quaternary structures. (A) Neutralizing potency of various-sized antibodies against Victoria virus was defined as the MNC of antibodies (IgM + IgG + IgA) in each fraction. (B) Proportions of IgM, IgG, and IgA quantity (wt/wt) in each quaternary structure to total antibody quantity in each quaternary structure (IgM + IgG + IgA, weight). The mean MNC of each quaternary structure of antibody against Victoria virus (C) and NY virus (D) is shown. (E) Mean MNC of each quaternary structure of antibody against Indonesia virus (H5N1) from subjects vaccinated with H5N1 vaccines. (F) Proportion of IgA quantity (wt/wt) in each quaternary structure to total nasal IgA (weight). (G) Binding activities of IgA with various quaternary structures specific for HA of Victoria virus were defined as the MBC of IgA in each fraction. The mean MBC of each quaternary structure of IgA specific for HA of Victoria virus (H) and NY virus (I) is shown. All error bars are SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Poly, polymer; Tetra, tetramer; Tri, trimer.

Fig. S5.

Comparison of neutralizing potency against H5N1 virus between Igs with different quaternary structures. (A) BN/PAGE analysis of each fraction of gel-filtered nasal IgA. Five micrograms of Ig was applied in each lane. Bands were visualized by Coomassie staining. (B) Proportion of IgM, IgG, or IgA quantity (wt/wt) in each quaternary structure to quantity of total antibodies in each quaternary structure (IgM + IgG + IgA, weight). The number of subjects is six. Error bars represent SEM. (C) Neutralizing potencies of variously sized antibodies against A/Indonesia/5/2005 (H5N1) virus were defined as MNCs of antibodies (IgM + IgG + IgA) in each fraction. The bar graph indicates the mean MNCs of antibodies in each individual. The number of subjects is six. Error bars represent SEM. The MNC of antibody against A/Indonesia/5/2005 (H5N1) virus (vaccination strain) in each fraction progressively decreased as molecular size increased. Poly, polymer; Tetra, tetramer; Tri, trimer.