Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013-2014 influenza season

Abstract

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013-2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013-2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.

Keywords: antibody; antigenic drift; hemagglutinin; influenza; vaccine.

Fig. 1.

pH1N1 viruses rapidly acquired HA mutation K166Q during the 2013–2014 influenza season. (A) Residue K166 (red) is shown on the A/California/04/2009 HA trimer [PDB ID code 3UBN (6)]. (B) Plotted is the frequency of different amino acid identities at HA residue 166 in pH1N1 HA sequences as a function of time. Nearly all pH1N1 possessed K166 from 2009 to mid-2012, but most isolates possessed Q166 by the 2013–2014 season. (C) A phylogenetic tree of pH1N1 viruses with branches colored according to amino acid identity at site 166 illustrates the rapid fixation of K166Q in recent pH1N1 isolates.

Fig. 2.

Adult humans possess Abs that bind to a region of HA that was recently mutated in pH1N1. (A) Sera were isolated from healthy donors (n = 195) from the state of New York during the 2013–2014 influenza season. HAI assays were performed using viruses with either WT A/California/07/2009 HA or A/California/07/2009 HA with a K166Q HA mutation. For each sera sample, we completed three independent HAI assays. Raw HAI data are reported in Table S1. Percentages of samples that had at least a twofold reduction in HAI titer using the mutant virus in three independent experiments are shown. K166-specificity of sera from individuals born between 1965 and 1979 is statistically significant compared with K166-specificity of sera from individuals born after 1985 (Fisher’s exact test; *P < 0.0001). (B) Homology between the A/Chile/01/1983 sH1 and the A/California/04/2009 pH1 are shown using the crystal structure of the A/California/04/2009 HA [PDB ID code 3UBN (6)]. Residue K166 is colored green. Amino acids that differ between A/Chile/01/1983 and A/California/04/2009 are shown in red. The glycan receptor is shown in black.

Fig. 3.

Vaccination of middle-aged adults with the current pH1N1 vaccine strain elicits Abs that bind to a region of HA that is now mutated in most pH1N1 isolates. (A) Healthy adult volunteers were vaccinated with a monovalent pH1N1 vaccine in 2009. Sera were isolated prevaccination and 30 d postvaccination and HAI assays were performed using viruses with either WT A/California/07/2009 HA or A/California/07/2009 HA with a K166Q HA mutation. Shown are HAI titers for donors that possessed K166 HA-specific Abs following vaccination. Data are representative of three independent experiments. Raw HAI titers for all donors are shown in Table S4. (B) ELISAs were completed using mAbs isolated from healthy adult volunteers that were vaccinated with a monovalent pH1N1 vaccine in 2009. ELISAs were coated either with A/California/07/2009 (WT) or A/California/07/2009 with a K166Q HA mutation. Shown are percentage of mAbs that bound to both viruses and percentage of mAbs that bound to the WT virus but not the mutant virus (n = 42 mAbs). Data are representative of two independent experiments. (C) A K166 HA-specific mAb (SFV009-3F05) or a mAb that recognizes both WT and K166Q-HA pH1N1 (SFV015-1F02) were injected into BALB/c mice (n = 4 per group). Twelve hours later, mice were then infected with 20,000 TCID₅₀ of WT or K166Q-HA virus and weight loss and survival were recorded for 11 d. Data are representative of two independent experiments.

Fig. 4.

Ferrets sequentially infected with A/Chile/01/1983 and A/California/07/2009 develop K166 HA-specific Abs. Ferrets were infected with a sH1N1 virus and then reinfected 84 d later with the A/California/07/2009 pH1N1 virus. Sera were collected 14 d after the second infection and HAI assays were completed using WT and K166Q-HA pH1N1 viruses. Shown are percentages of samples that had at least a twofold reduction in HAI titer using the K166Q HA mutant virus in three independent experiments. Raw HAI titers are shown in Table S5. The difference in K166 HA-specificity is statistically significant comparing the A/Chile/01/1983-A/California/07/2009 group with the rest of the groups (3 of 8 vs. 0 of 22; Fisher’s exact test P < 0.05).