Imprinted Anti-Hemagglutinin and Anti-Neuraminidase Antibody Responses after Childhood Infections of A(H1N1) and A(H1N1)pdm09 Influenza Viruses

Abstract

Immune imprinting is a driver known to shape the anti-hemagglutinin (HA) antibody landscape of individuals born within the same birth cohort. With the HA and neuraminidase (NA) proteins evolving at different rates under immune selection pressures, anti-HA and anti-NA antibody responses since childhood influenza virus infections have not been evaluated in parallel at the individual level. This is partly due to the limited knowledge of changes in NA antigenicity, as seasonal influenza vaccines have focused on generating neutralizing anti-HA antibodies against HA antigenic variants. Here, we systematically characterized the NA antigenic variants of seasonal A(H1N1) viruses from 1977 to 1991 and completed the antigenic profile of N1 NAs from 1977 to 2015. We identified that NA proteins of A/USSR/90/77, A/Singapore/06/86, and A/Texas/36/91 were antigenically distinct and mapped N386K as a key determinant of the NA antigenic change from A/USSR/90/77 to A/Singapore/06/86. With comprehensive panels of HA and NA antigenic variants of A(H1N1) and A(H1N1)pdm09 viruses, we determined hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) antibodies from 130 subjects born between 1950 and 2015. Age-dependent imprinting was observed for both anti-HA and anti-NA antibodies, with the peak HI and NI titers predominantly detected from subjects at 4 to 12 years old during the year of initial virus isolation, except the age-independent anti-HA antibody response against A(H1N1)pdm09 viruses. More participants possessed antibodies that reacted to multiple antigenically distinct NA proteins than those with antibodies that reacted to multiple antigenically distinct HA proteins. Our results support the need to include NA proteins in seasonal influenza vaccine preparations. IMPORTANCE Seasonal influenza vaccines have aimed to generate neutralizing anti-HA antibodies for protection since licensure. More recently, anti-NA antibodies have been established as an additional correlate of protection. While HA and NA antigenic changes occurred discordantly, the anti-HA and anti-NA antibody profiles have rarely been analyzed in parallel at the individual level, due to the limited knowledge on NA antigenic changes. By characterizing NA antigenic changes of A(H1N1) viruses, we determined the anti-HA and anti-NA antibody landscape against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses using sera of 130 subjects born between 1950 and 2015. We observed age-dependent imprinting of both anti-HA and anti-NA antibodies against strains circulated during the first decade of life. A total of 67.7% (88/130) and 90% (117/130) of participants developed cross-reactive antibodies to multiple HA and NA antigens at titers ≥1:40. With slower NA antigenic changes and cross-reactive anti-NA antibody responses, including NA protein in influenza vaccine preparation may enhance vaccine efficacy.

Keywords: antigenic drift; hemagglutinin; imprinting; influenza; neuraminidase.

FIG 1

Identification of NA antigenic variants from A(H1N1) viruses circulating between 1977 and 1991. NI titers were determined using ferret antisera and recombinant H6N1 viruses carrying NA protein derived from (A) USSR/77, (B) Chile/83, (C) Singapore/86, or (D) Texas/91. (E) Antigenic cartography map was generated using the endpoint NI titers, as shown in Table 1. NI titer values of 5 were assigned for all results below the ELLA limit-of-detection (NI titer <10). Recombinant H6N1 viruses (filled circle icons) and H1N1-raised ferret antisera (open square icons) are color-coded by N1 genetic similarity to USSR/77 (green), Chile/83 (CHL/83; orange), Singapore/86 (SGP/86; purple), and Texas/91 (TX/91; blue). Grid increments between two icons indicate a 2-fold difference in modeled NI titers with two units corresponding to a 4-fold reduction, three units to an 8-fold reduction, etc.

FIG 2

Mapping amino acid changes responsible for the antigenic drift from USSR/77 to Singapore/86. (A) The NA enzyme active site and key amino acid changes, S247N, K369R, N386K, and K/R434N were mapped on the N1 tetramer of A/Vietnam/1203/2004 (PDB 2HU0) using PyMOL. (B) Magnified view of N1 monomer of A/Vietnam/1203/2004 (PDB 2HU0) with the active site residues highlighted in orange and critical amino acid residues highlighted in pink. (C) Reactivity of anti-USSR/77 ferret sera against NA protein derived from USSR/77, Singapore/86, or USSR/77 with different amino acid changes. (D) Reactivity of anti-Singapore/86 ferret sera against NA protein derived from USSR/77, Singapore/86, or USSR/77 with different amino acid changes. (E) Reactivity of anti-USSR/77 ferret sera against NA protein derived from USSR/77, Singapore/86, or Singapore/86 with K386N substitution. (F) Reactivity of anti-Singapore/86 ferret sera against NA protein derived from USSR/77, Singapore/86, or Singapore/86 with K386N substitution.

FIG 3

Phylogenetic tree of N1 sequences from seasonal A(H1N1) circulated from 1977 to 2008. A phylogenetic tree was generated using the full coding regions of the NA proteins from representative strains from 1977 to 2008. The strains are color-coded depending on the amino acid substitution on residue 386. The viruses with amino acid substitution of serine (S), asparagine (N), lysine (K), and aspartic acid (D) at residue 386 are shown in blue, green, orange, and pink, respectively.

FIG 4

Age-dependent HI and NI antibody titers to antigenically distinct H1 HA and N1 NA of A(H1N1) and A(H1N1)pdm09 viruses from 1977 to 2015. (A) A heatmap of 130 individuals’ HI titers against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses. The HI titers of each individual were represented in a row, sorted according to the birth year, with the youngest subjects shown on the top and the oldest subjects at the bottom. HI titers were shown in the log₂ scale, with the light-yellow shading indicating a higher HI titer and the black shading indicating a negative result (HI<1:10). (B) Generalized Additive Model (GAM) was fitted to the HI titers measured against A(H1N1) and A(H1N1)pdm09 viruses. The x axis represents the age of each individual at the year of virus isolation of each A(H1N1) and A(H1N1)pdm09 viruses. The y axis represents the HI titer. The fitted lines represent mean log₂ HI titers against each A(H1N1) and A(H1N1)pdm09 viruses and are color coded by the virus strain. Individuals showing HI titer <1:10 are arbitrarily assigned the titer 5. (C) A heatmap generated using 130 individuals’ NI titers against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses. (D) GAM was fitted to the NI titers measured against A(H1N1) and A(H1N1)pdm09 viruses. The x axis represents the age of each individual at the year of virus isolation of each A(H1N1) and A(H1N1)pdm09 viruses. Individuals showing HI or NI titer <1:10 are arbitrarily assigned the titer 5.