Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin

Abstract

Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for "original antigenic sin." Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional neuraminidase inhibition titers. This is an important finding since ELISA is a simpler method than functional assays and can be implemented in high-throughput settings to analyze large numbers of samples. Furthermore, we showed that low titers of broadly cross-reactive antibodies against neuraminidase are prevalent in humans. By the use of an appropriate vaccination strategy, these titers could potentially be boosted to levels that might provide broad protection from influenza virus infection.

Keywords: influenza; influenza virus; neuraminidase; neuraminidase inhibition.

Copyright © 2017 Rajendran et al.

Figures

FIG 1

Anti-NA titers against human and avian influenza virus isolates in serum of children (6 to 59 months of age), adults (18 to 49 years of age), and the elderly (≥65 years of age). (A to C) Mean baseline anti-NA ELISA titers of the three different age groups were visualized using heat maps for (A) N1 (SC18, USSR77, NC99, Cal09, and VN04), (B) N2 (Sing57, HK68, Phil82, Pan99, and ck97), and (C) influenza B virus NA (NB; Lee40, Yam88, Mal04, Flor06, and Wisc10). *, pandemic strain. (D to F) Serum reactivity profiles of the three age cohorts against (D) N1, (E) N2, and (F) influenza B virus NA spanning the years 1918 to 2014. Serum reactivity profiles are shown using geometric mean titers, with error bars displaying standard errors of the means. The dashed lines correspond to 2006/2008, indicating the years during which the serum samples were collected. AUC, area under the curve. (G to I) Phylogenetic trees of influenza virus neuraminidase proteins used in this study: (G) N1; (H) N2; (I) influenza B virus NA (NB). Scale bars represent a 7% difference in amino acid identity. The trees depicted in panels G to I were rooted using the sequence of the N10 NA-like protein (A/little yellow-shouldered bat/Guatemala/060/2010).

FIG 2

Relationship between binding and functionality of antibodies against prepandemic seasonal N1, N2, and type B influenza virus NA. (A and B) Geometric mean titers for children, adults, and the elderly against recombinant NC99 N1 as measured by ELISA (prevaccination, shown as the area under the curve [AUC]) (A) and ELLA titers (pre- and postvaccination, shown as IC50 values) against H6N1NC99 virus (B). The dashed line indicates the starting serum dilutions used in the ELLA assays. (C) Correlation between NC99 N1 ELLA and ELISA titers (Spearman r = 0.5549, P < 0.0001). (D and E) The same analyses were also performed for anti-N2 antibodies with recombinant Pan99 N2 as an ELISA substrate (pre- and postvaccination) (D) and H6N2Pan99 as an ELLA reagent (pre- and postvaccination) (E). D, day. (F) Similarly to the data determined with N1, the binding and functional titers against Pan99 N2 correlated well (Spearman r = 0.6981, P < 0.0001). (G) Design of the H6NBYam88 virus that was rescued specifically for this study. The virus is a reassortant virus that combines the internal genes of H1N1 virus A/PR/8/34, an H6 HA, and a chimeric NA. The NA consists of the ectodomain of the B/Yamagata/16/1988 NA combined with the cytoplasmic tail and transmembrane domains from the A/PR/8/34 N1 NA. The open reading frame is flanked by 5′ and 3′ noncoding regions (NCRs) of the A/PR/8/34 NA. (H and I) Geometric mean titers for children, adults, and the elderly against recombinant Yam88 NB as measured by ELISA (prevaccination) (H); ELLA titers (pre- and postvaccination) against the novel H6NBYam88 virus (I). (J) Relationship between binding and functional Yam88 NA titers (Spearman r = 0.8643, P < 0.0001).

FIG 3

Binding and functionality of antibodies against the 2009 pandemic H1N1 NA. (A) Geometric mean Cal09 N1 ELISA titers (AUC) of sera from children, adults, and the elderly (prepandemic) and sera from adults collected after the 2009 pandemic. The postpandemic adult sera had significantly higher Cal09 N1 ELISA titers than the sera from the three age groups that were not exposed to the 2009 pandemic virus. (B) Mean ELLA titers against the H6N1Cal09 virus before and after vaccination. The dashed line indicates the starting serum dilutions used in the ELLA assays. (C) The Ella IC50 H6N1Cal09 values and the Cal09 N1 ELISA titers in sera from individuals not exposed to the 2009 pandemic H1N1 virus do not correlate (Spearman r = 0.09914). n.s., not significant. (D) Titers from postpandemic sera do correlate well (Spearman r = 0.5592, P = 0.0001). (E) Mean H6 HA ELISA titers of the prepandemic cohort (children, adults, and the elderly; collected on day 0). The elderly had high baseline titers compared to children and adults. (F) The ELLA IC50s for H6N1Cal09 correlate well with the ELISA titers measured against H6 for individuals not exposed to 2009 H1N1 pandemic virus (Spearman r = 0.726, P < 0.0001).

FIG 4

Anti-HA stalk antibodies interfere with NI activity in ELLA assays. (A) ELLA IC50 values for broadly reactive pan-HA stalk MAb CR9114 against H6NX and wild-type viruses. (B) ELLA IC50 values for broadly reactive group 1 MAb KB2 as measured with H6NX and wild-type viruses. (C) NI against H3N2 viruses in the presence of H3 stalk-reactive MAb 12D1. (D to I) Representative NI data from various H6NX and wild-type viruses in the presence of MAb CR9114, MAb KB2, or MAb 12D1 or an IgG negative-control MAb were plotted and fitted to nonlinear regression curves. (J and K) The data shown explain the potential mechanism of anti-HA stalk antibodies interfering with NI activity. (J) Anti-NA antibodies robustly interfere with neuraminidase activity by directly binding to NA and inhibition/shielding of the enzymatic site. (K) Anti-HA stalk antibodies bind to membrane-proximal sites on the HA and, via steric hindrance, indirectly shield the enzymatic site of NA and thus interfere with neuraminidase activity. However, this interference seems less robust than the inhibition from anti-NA antibodies.