IgA polymerization contributes to efficient virus neutralization on human upper respiratory mucosa after intranasal inactivated influenza vaccine administration

Abstract

Unlike the current injectable influenza vaccines, intranasally administered influenza vaccines induce influenza virus-specific IgA antibodies in the local respiratory mucosa as well as IgG antibodies in the systemic circulation. Our previous study showed that after five volunteers underwent intranasal administration with inactivated H3N2 or H5N1 vaccines, their IgA antibodies on the upper respiratory tract were present as monomers, dimers, and multimers (trimers and tetramers). Moreover, the multimers associated with the highest virus neutralizing activity. However, it has remained elusive whether a more practical intranasal vaccination strategy could induce the high-performance IgA multimers in the nasal mucosa. In the present study, volunteers were administered with two doses of the intranasal trivalent whole-virus inactivated influenza vaccine and showed that in nasal wash samples the amount of multimeric IgA correlated positively with virus neutralizing titers, indicating that the multimeric IgA antibodies play an important role in the antiviral activity at the nasal mucosa. Surface plasmon resonance analysis of the binding dynamics of nasal wash derived IgA monomers, dimers, and multimers against recombinant trimeric influenza virus HA showed that sample fractions containing IgA multimers dissociated from HA less well than sample fractions without IgA multimers. Thus, IgA multimers may "stick" to the antigen more tightly than the other structures. In summary, intranasal administration of two doses of multivalent inactivated influenza vaccines induced multimeric IgA. Multimerization of mucosal IgA antibodies conferred higher neutralizing activity against viruses in the nasal mucosa, possibly by increasing their cohesion to virus antigens.

Keywords: Secretory IgA antibody; influenza virus; intranasal inactivated influenza vaccine; multimeric SIgA antibody; upper respiratory mucosa.

Figure 1.

Neutralizing test (NT) titers against four influenza virus strains in (A) serum and (B) nasal wash samples collected from 47 healthy adult volunteers before (Pre) and 3 (3W) and 6 weeks (6W) after intranasal vaccination. A/Tx(H3N2): A/Texas/50/2012 (H3N2) (a vaccine strain); A/Vic(H3N2): A/Victoria/361/2011 (H3N2) (the vaccine strain from the previous year); A/Cal(H1N1): A/California/7/2009 (H1N1)pdm09 (a vaccine strain); B/Mc (Yam): B/Massachusetts/2/2012 (Yamagata-lineage) (a vaccine strain). Each dot represents the NT titer of the individual volunteer-derived samples. The data are expressed as scatter plots with mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001, as determined by One-way ANOVA. (C) The correlation between serum and nasal wash neutralizing test (NT) titers at 6 week against various virus strains were examined using Pearson's correlation coefficient (r).

Figure 2.

Biochemical characterization of the human nasal wash-derived IgA samples. (A) Relative proportions of IgA, IgG, and IgM in the total immunoglobulin of the concentrated nasal washes collected from 21 individuals 3 weeks after the second vaccination. These 21 subjects were selected from the original cohort of 47 because they provided large amounts of wash samples. Each dot represents an individual volunteer-derived sample. (B) The purified nasal wash IgA samples of the 21 individuals were subjected to size exclusion chromatography and fractionated into fractions Fr1, Fr2, and Fr3 based on the three peaks on the chromatogram. A representative chromatogram is shown. (C) Relative proportions of the total nasal IgA that is contained in the Fr1, Fr2, and Fr3 fractions. (D) Blue native-PAGE analysis of the Fr1, Fr2, and Fr3 fractions from three representative individuals (#03, #07, and #08). Arrowheads indicate monomers and dimers while the square bracket indicates multimers. (E) IgA2/IgA1 ratio in the Fr1, Fr2, and Fr3 fractions from the 21 individuals, as determined by IgA subunit quantification by LC-MS. (F) J chain (JC)/ IgA heavy chain (HC) ratio of the Fr1, Fr2, and Fr3 fractions of the 21 volunteers, as determined by IgA subunit quantification by LC-MS. Dotted lines indicate the theoretical JC/HC values of the dimers (0.25), trimers (0.167), and tetramers (0.125). The data are expressed as scatter plots with the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by One-way ANOVA.

Figure 3.

Correlation analyses assessing the relationship between serum and nasal wash neutralizing test (NT) titers against various virus strains and (A) the proportion of total nasal immunoglobulin (Ig) that is IgG, (B) the proportion of total nasal Ig that is IgA, (C) the proportion of total nasal IgA that is in Fr1, (D) the proportion of total nasal IgA that is in Fr2, and (E) the proportion of total nasal IgA that is in Fr3. Each dot represents an IgA sample from each of the 21 subjects who provided large amounts of nasal wash sample. The viruses that were tested were A/California/7/2009 (H1N1)pdm09 (green, A/Cal(H1N1)), A/Victoria/210/2009 (H3N2) (blue, A/Vic(H3N2)), A/Texas/50/2012 (H3N2) (red, A/Tx(H3N2)), and B/Massachusetts/2/2012 (Yamagata-lineage) (black, B/Mc (Yam)). The only positive correlation was that between the proportion of total nasal IgA that is in Fr3 and the nasal wash NT titers against A/Victoria/361/2011 (H3N2).

Figure 4.

Binding dynamics of human nasal IgA samples to HA. (A) Relative proportions of IgA monomers, dimers, and multimers in the Fr1, Fr2, and Fr3 fractions from three representative individuals (#03, #07, and #08). These calculations were performed on the basis of density quantification of the bands on blue native-PAGE gels (see Fig. 2D). (B) These Fr1, Fr2, and Fr3 IgA samples were subjected to surface plasmon resonance analysis of their binding dynamics to the recombinant trimeric hemagglutinin from A/Victoria/361/2011 (H3N2). Sensorgrams were y-axis adjusted (0 = baseline, 100 = binding) to allow comparisons between samples in terms of the dissociation rate of IgA from HA. (C) Fr1, Fr2 and Fr3 IgA samples derived from 10 volunteers were subjected to SPR analysis. Dissociation efficiency of Fr2 and Fr3, which were defined as the reciprocal of area under the dissociation curve (AUC) of SPR response, were significantly lower than that of Fr1. The data are expressed as scatter plots with mean ± SD. *p < 0.05, **p < 0.01, as determined by paired t-test. ns, not significant.