Immune history shapes specificity of pandemic H1N1 influenza antibody responses

Abstract

Human antibody responses against the 2009 pandemic H1N1 (pH1N1) virus are predominantly directed against conserved epitopes in the stalk and receptor-binding domain of the hemagglutinin (HA) protein. This is in stark contrast to pH1N1 antibody responses generated in ferrets, which are focused on the variable Sa antigenic site of HA. Here, we show that most humans born between 1983 and 1996 elicited pH1N1 antibody responses that are directed against an epitope near the HA receptor-binding domain. Importantly, most individuals born before 1983 or after 1996 did not elicit pH1N1 antibodies to this HA epitope. The HAs of most seasonal H1N1 (sH1N1) viruses that circulated between 1983 and 1996 possess a critical K133 amino acid in this HA epitope, whereas this amino acid is either mutated or deleted in most sH1N1 viruses circulating before 1983 or after 1996. We sequentially infected ferrets with a 1991 sH1N1 virus and then a pH1N1 virus. Sera isolated from these animals were directed against the HA epitope involving amino acid K133. These data suggest that the specificity of pH1N1 antibody responses can be shifted to epitopes near the HA receptor-binding domain after sequential infections with sH1N1 and pH1N1 viruses that share homology in this region.

Figure 1.

Modeling and electrostatics of pH1N1 HA mutants. The structures and electrostatic potentials of pH1N1 HAs with G158E or K133N mutants were modeled. Shown are pH1N1-WT (A) and computationally modeled pH1N1 HAs with G158E (B) or K133N (C) mutations. Sialic acid is shown as a stick structure. Surface coloring indicates electrostatic potential in units of kT/e where k is Boltzmann’s constant, T is temperature, and e is the elementary charge.

Figure 2.

pH1N1 antibody responses in previously naive ferrets are dominated against Sa antigenic site of HA. (A) HAI assays were completed using viruses possessing either wild-type A/California/07/2009 HA or A/California/07/2009 HA with G158E or K133N mutations. HAI assays were performed with sera isolated from ferrets 14 d after infection with the A/California/07/2009 pH1N1 strain. Fold change for each mutant virus (WT HAI titer/mutant HAI titer) is shown here. Each triangle represents an individual sera sample, and the mean is indicated with a line. HAI titers using the G158E mutant virus are lower compared with HAI titers using the WT virus, as determined using a paired Student’s t test on log₂ transformed data (*, P = 0.016). Data are representative of three independent experiments. (B) Direct flow cytometry-based antibody (Ab) binding assays were completed using pooled anti-sera. pH1N1-infected MDCK cells were incubated with ferret anti-sera, and antibody binding was determined after addition of a FITC anti-ferret antibody. Data are representative of three independent experiments.

Figure 3.

pH1N1 antibody responses in humans born between 1983–1996 are dominated against region of HA involving K133. (A–C) HAI assays were completed using viruses possessing either wild-type A/California/07/2009 HA or A/California/07/2009 HA with G158E or K133N mutations. HAI assays were performed with sera isolated from humans 9–31 d after onset of pH1N1 symptoms. Humans were naturally infected with pH1N1 (PCR-verified). HAI titers are shown in Table S3. Fold change for each mutant virus (WT HAI titer/mutant HAI titer) is shown here. Each triangle represents an individual sera sample, and the mean is indicated with a line. Individuals are separated based on year of birth. 7 of 8 sera samples from individuals born between 1983 and 1996 had reduced titers to the K133N mutant, whereas only 2 sera samples from the other time periods (n = 46) had reduced titers to the K133N mutant (K133-specificity of sera from 1983–1996 is significantly different compared with sera from other time periods; Fisher’s exact test; *, P < 0.001). HAI data are representative of three independent experiments. (D) Timeline depicting the percentage of sH1N1 isolates possessing Lys (solid line) or a mutation/deletion (dotted line) at position 133 of HA based on sequences contained in the NCBI database. In total, 7,045 sequences resulting from unique isolates were aligned using the program MUSCLE to yield K133 prevalence on a yearly basis. Isolates from 1918–1957 were grouped due to low number of sequences from these years. sH1N1 viruses did not circulate between 1958 and 1976, and this is indicated by a dashed vertical line.

Figure 4.

Human monoclonal antibodies specific for pH1N1 HA epitope involving aa 133 react strongly with a 1991 sH1N1 strain. HAI assays were completed using monoclonal antibodies derived from pH1N1-infected humans. HAI assays were completed using viruses possessing either wild-type pH1N1 HA (A/California/07/2009), pH1N1 with a G158E HA mutation, pH1N1 with a K133N HA mutation, or sH1N1 viruses from 1957 (A/Denver/1/1957), 1991 (A/Texas/36/1991), or 2007 (A/Brisbane/59/2007). Shown are minimum amounts of antibody required to inhibit agglutination of red blood cells in the HAI assay. Data are representative of three independent experiments.

Figure 5.

sH1N1 preexposure alters the specificity of pH1N1 antibody responses in ferrets. (A–D) HAI assays were completed using viruses possessing either wild-type A/California/07/2009 HA, or A/California/07/2009 HA with G158E or K133N mutations. Sera were isolated from ferrets sequentially infected with a sH1N1 strain, and then the A/California/07/2009 pH1N1 strain. Infections were completed 84 d apart and sera were isolated 14 d after infection with the pH1N1 virus. Fold change for each mutant virus (WT HAI titer/mutant HAI titer) is shown here. Each triangle represents an individual sera sample, and the mean is indicated with a line. Differences in K133 specificity were statistically significant between the different preexposed groups (Fisher’s exact test; *, P = 0.003 comparing HAI titers of sera isolated from A/Texas/36/1991 preexposed animals to HAI titers of sera isolated from animals preexposed with other sH1N1 viruses). Data are representative of two independent experiments.