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

Significance

Influenza viruses typically cause a higher disease burden in children and the elderly, who have weaker immune systems. During the 2013–2014 influenza season, H1N1 viruses caused an unusually high level of disease in middle-aged adults. Here, we show that recent H1N1 strains possess a mutation that allows viruses to avoid immune responses elicited in middle-aged adults. We show that current vaccine strains elicit immune responses that are predicted to be less effective in some middle-aged adults. We suggest that new viral strains should be incorporated into seasonal influenza vaccines so that proper immunity is elicited in all humans, regardless of age and pre-exposure histories.

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.

Seasonal H1N1 (sH1N1) viruses circulated in the human population for much of the last century and, as of 2009, most humans had been exposed to sH1N1 strains. In 2009, an antigenically distinct H1N1 strain began infecting humans and caused a pandemic (1–3). Elderly individuals were less susceptible to 2009 pandemic H1N1 (pH1N1) viruses because of cross-reactive antibodies (Abs) elicited by infections with older sH1N1 strains (3–7). pH1N1 viruses have continued to circulate on a seasonal basis since 2009. Influenza viruses typically cause a higher disease burden in children and elderly individuals (8) but pH1N1 viruses caused unusually high levels of disease in middle-aged adults during the 2013–2014 influenza season (9–12). For example, a significantly higher proportion of individuals aged 30- to 59-y-old were hospitalized in Mexico with laboratory-confirmed pH1N1 cases in 2013–2014 relative to 2011–2012 (11).

Most neutralizing influenza Abs are directed against the hemagglutinin (HA) glycoprotein. International surveillance laboratories rely primarily on ferret anti-influenza sera for detecting HA antigenic changes (13). For these assays, sera are isolated from ferrets recovering from primary influenza infections. Seasonal vaccine strains are typically updated when human influenza viruses acquire HA mutations that prevent the binding of primary ferret anti-influenza sera. Our laboratory and others have demonstrated that sera isolated from ferrets recovering from primary pH1N1 infections are dominated by Abs that recognize an epitope involving residues 156, 157, and 158 of the Sa HA antigenic site (14, 15). The pH1N1 component of the seasonal influenza vaccine has not been updated since 2009 because very few pH1N1 isolates possess mutations in residues 156, 157, and 158. The majority of isolates from the 2013–2014 season have been labeled as antigenically similar to the A/California/07/2009 vaccine strain (9).

It is potentially problematic that major antigenic changes of influenza viruses are mainly determined using antisera isolated from ferrets recovering from primary influenza infections. Unlike experimental ferrets, humans are typically reinfected with antigenically distinct influenza strains throughout their life (16). In the 1950s, it was noted that the human immune system preferentially mounts Ab responses that cross-react to previously circulating influenza strains, as opposed to new Ab responses that exclusively target newer viral strains (17). This process, which Thomas Francis Jr. termed “original antigenic sin,” has been experimentally recapitulated in ferrets (14, 18), mice (19–21), and rabbits (22). Our group and others recently demonstrated that the specificity of pH1N1 Ab responses can be shaped by prior sH1N1 exposures (14, 23–26). We found that ferrets sequentially infected with sH1N1 and pH1N1 viruses mount Ab responses dominated against epitopes that are conserved between the viral strains (14). These studies indicate that primary ferret antisera may not be fully representative of human influenza immunity.

It has been proposed that increased morbidity and mortality of middle-aged adults during the 2013–2014 influenza season is primarily a result of low vaccination rates within these populations (27). An alternative explanation is that recent pH1N1 strains have acquired a true antigenic mutation that has been mislabeled as “antigenically neutral” by assays that rely on primary ferret antisera. Here we complete a series of experiments to determine if recent pH1N1 strains possess a mutation that prevents binding of Abs in middle-aged humans who have been previously exposed to different H1N1 strains.

Results

Recent pH1N1 Strains Possess a Mutation That Prevents Binding of Human Antibodies.

Antisera isolated from ferrets recovering from primary pH1N1 infections are highly specific for an epitope involving residues 156, 157, and 158 of the Sa HA antigenic site (14, 15). Very few pH1N1 isolates possess mutations in these Sa residues (Fig. S1); however, pH1N1 viruses recently acquired a K166Q HA mutation, which is located at the interface of the Sa/Ca (28) antigenic sites (Fig. 1A). The K166Q HA mutation first arose during the 2012–2013 season and is now present in over 99% of pH1N1 isolates (Fig. 1 B and C). Based on experiments using primary antisera isolated from infected ferrets, surveillance laboratories have reported that pH1N1 viruses with the K166Q HA mutation are antigenically indistinguishable from the A/California/7/2009 pH1N1 vaccine strain (9).

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.

To address if human Abs are capable of recognizing pH1N1 viruses with the K166Q HA mutation, we performed hemagglutination-inhibition (HAI) assays using sera from healthy humans collected during the 2013–2014 influenza season in the United States. Remarkably, 27% of sera from individuals born between 1940 and 1984 possessed Abs specific for an epitope involving K166 (Fig. 2A and Table S1). Over 42% of individuals born from 1965 to 1979 had K166 HA-specific Abs in their sera (n = 54 individuals). Sera isolated from individuals born between 1985 and 1997 (n = 49 individuals) did not have detectable levels of K166 HA-specific Abs. Differences in K166 HA-specificity were statistically significant between sera isolated from individuals born between 1965 and 1979 and individuals born after 1985 (Fisher’s exact test; P < 0.0001). Similar results were obtained when we analyzed sera from healthy humans collected during the 2013–2014 influenza season in Mexico (Fig. S2 and Table S2).

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.

It is remarkable that HAI assays, which are relatively insensitive, were able to reproducibly detect K166-specific Abs in so many individuals in our experiments. Fig. 2A and Fig. S2 show percentages of donors that had at least a twofold reduction in HAI titer using the K166Q HA mutant virus in three independent assays. It is worth pointing out that many sera samples had over fourfold reduced HAI titers using a pH1N1 virus engineered to possess the single K166Q HA mutation compared with the pH1N1 vaccine strain (Table S1). Age-related differences in K166 HA-specificity among United States donors remained statistically significant using a fourfold reduction in HAI titer as a cut-off (Fisher’s exact test; P < 0.05 comparing donors born between 1965 and 1979 and individuals born after 1985). Sera that had K166 HA-specificity based on HAI assays failed to efficiently neutralize K166Q-possessing viruses in in vitro neutralization assays (Table S3). K166 HA-specific sera were also unable to recognize a primary viral isolate collected in 2013 (A/CHOP/1/2013) that possesses a K166Q HA mutation (Table S3).

The K166 Epitope Is Shielded by a Glycosylation Site Present in sH1N1 Viruses Circulating After 1985.

Original antigenic sin Abs are originally primed by influenza strains that circulated in the past (14, 17, 18, 22, 29). We propose that K166 HA-specific Ab responses were likely primed by sH1N1 viruses circulating in humans before 1985 and then boosted by the 2009 pH1N1 virus. Sera isolated from individuals born between 1965 and 1979 had the highest K166 HA-specificity (both in percent and titer) (Fig. 2A and Table S1). sH1N1 viruses that circulated in the late 1970s and early 1980s share extensive homology with pH1N1 viruses in the vicinity of K166 (Fig. 2B). sH1N1 viruses were absent from the human population from 1957 to 1976 and began infecting humans again in 1977. Therefore, humans born between 1957 and 1976 likely had their first H1N1 encounter with a sH1N1 virus that shared homology with pH1N1 viruses in the vicinity of K166.

In 1986, sH1N1 viruses acquired a new glycosylation site at HA amino acid 129 that is predicted to shield the epitope involving K166 (Figs. S3 and S4). The absence of K166 HA-specific responses in individuals born after 1985 is likely because sH1N1 viruses glycosylated at HA amino acid 129 fail to prime K166 HA-specific responses. The lower number of K166 HA-specific responders born in the 1950s might also be attributed to unique glycosylation sites in sH1N1 viruses that circulated during this time period (Fig. S3), although precise glycosylation statuses of viruses circulating before 1977 are uncertain because of limited numbers of sequenced viruses. Although we did not examine sera from very elderly individuals, it is possible that they also have immunodominant K166 HA-specific responses, because a recent study reported that a mAb isolated from a survivor of the 1918 H1N1 pandemic binds to pH1N1 in an epitope involving K166 (6). There is considerable homology between the 1918 H1N1 and the 2009 H1N1 in the vicinity of K166 (6).

To experimentally address if glycosylation sites present in previous sH1N1 strains shield the epitope involving K166, we used reverse-genetics to produce pH1N1 viruses that had glycosylation sites that were either present in sH1N1 strains from 1977 to 1985 (sites 131+163) or 1986–2008 (sites 129+163). Western blot analysis revealed that residues 129 and 131, but not residue 163, were glycosylated in our reverse-genetics derived viruses (Fig. S4B). Consistent with the hypothesis that the K166 epitope is shielded by glycosylation sites present in 1986–2008 sH1N1 viruses, K166 HA-specific human sera had reduced titers to pH1N1 viruses with the 129 glycosylation site but normal titers to pH1N1 viruses with the 131 glycosylation site (Fig. S4C). As a control, we also completed HAI assays with sera from donors that were born in the 1970s who did not have detectable levels of K166 HA-specific sera Abs. As expected, these sera did not have reduced titers to pH1N1 viruses with the 129 glycosylation site, but interestingly, these sera did have reduced titers to pH1N1 viruses with the 131 glycosylation site (Fig. S4C). We previously demonstrated that Ab responses focused on an epitope near the 131 glycosylation site can be elicited by sequential infections with a sH1N1 virus from the early 1990s and the pH1N1 virus (14). We speculate that donors in the “non-K166 HA-specific” group were previously infected with antigenically distinct sH1N1 strains compared with donors in the K166 HA-specific group (ie: A/Singapore/06/1986-like strain vs. A/Chile/01/1983-like strain). Taken together, these data suggest that glycosylation sites on previously circulating sH1N1 viruses shield epitopes and influence the development of subsequent Ab responses against pH1N1 virus.

Vaccination with Current pH1N1 Vaccine Strain Elicits K166 HA-Specific Abs.

The pH1N1 vaccine strain has not been updated since 2009. We determined whether this vaccine strain, which possesses an HA that has K166, elicits K166 HA-specific Abs in humans. First, we analyzed sera from individuals vaccinated in 2009. All of the individuals in this cohort were born before 1984 and most did not have pH1N1 Ab titers before vaccination (Table S4). Sera from 5 of 17 individuals possessed detectable levels of K166 HA-specific Abs following vaccination (Fig. 3A and Table S4). Sera from all five of these individuals had <1:40 HAI titers against the K166Q HA mutant pH1N1 virus (Table S4). One K166 HA-specific individual (subject #1) possessed K166 HA-specific Abs before vaccination (Fig. 3A and Table S4). It is possible that this individual was naturally infected with pH1N1 before vaccination. All of the K166 HA-specific donors had detectable prevaccination Ab titers against sH1N1 viruses from 1977 and 1983; however, we also found titers against these strains in some donors that did not have detectable levels of K166 HA-specific serum Abs (Table S4). We also measured binding of 42 HA head-specific mAbs isolated from 12 adult donors (born 1949–1985) that were vaccinated against the pH1N1 strain in 2009. Strikingly, 23% of these mAbs had reduced binding to pH1N1 engineered to have the K166Q mutation (Fig. 3B). This finding is consistent with a previous report that identified several K166 HA-specific mAbs derived from a donor that was born before 1977 (30).

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.

We passively transferred a K166 HA-specific mAb (SFV009-3F05) or a control mAb that binds equally to WT and K166Q-HA pH1N1 viruses (SFV015-1F02) to BALB/c mice 12 h before infecting with a lethal dose of WT or K166Q-HA pH1N1 viruses. Control animals that did not receive a mAb before infection rapidly lost weight and died or needed to be euthanized (Fig. 3C). Mice receiving the control SFV015-1F02 mAb before infection with WT or K166Q-HA pH1N1 viruses all survived with minimal weight loss (Fig. 3C). Mice receiving the K166 HA-specific SFV009-3F05 mAb survived following infection with WT pH1N1 but rapidly lost weight and died or needed to be euthanized following infection with K166Q-HA pH1N1 (Fig. 3C). These data suggest that K166 HA-specific Abs can be less efficient at preventing disease in a mouse model following infection with a pH1N1 virus possessing K166Q HA.

Can K166 HA-Specific Immunity Be Recapitulated in Ferrets for Surveillance Purposes?

Current surveillance efforts rely heavily on antisera isolated from ferrets recovering from primary influenza virus infections. Ferret antisera could potentially be more reflective of human immunity if isolated from animals sequentially infected with antigenically distinct viral strains. We attempted to elicit K166 HA-specific Abs in ferrets by sequentially infecting animals with older sH1N1 strains and then the A/California/07/2009 pH1N1 strain.

We initially infected animals with a sH1N1 virus that circulated in 1977 (A/USSR/90/1977), a sH1N1 virus that circulated in 1983 (A/Chile/01/1983), or a sH1N1 virus that circulated in 1986 (A/Singapore/06/1986). After 84 d, we reinfected animals with the A/California/07/2009 pH1N1 strain. As controls, we infected some animals twice with A/California/07/2009 and other animals only once with A/California/07/2009. Three of eight of the ferrets sequentially infected with A/Chile/01/1983 and A/California/07/2009 mounted K166 HA-specific Abs detectable in HAI assays (Fig. 4 and Table S5). The 22 ferrets in the other experimental groups did not mount detectable levels of K166 HA-specific Abs. 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). K166 HA-specific Abs were likely not elicited in the A/Singapore/06/1986-A/California/07/2009 group because the K166 HA epitope is predicted to be shielded by a glycosylation site at residue 129 of A/Singapore/06/1986 (Fig. S4). It is interesting that K166 HA-specific Abs were not elicited by A/USSR/90/1977-A/California/07/2009 sequential infections. This result is likely because of variation at residue 125, which is close to residue 166 (Fig. S5). A/Chile/01/1983 and A/California/07/2009 both possess S125, whereas A/USSR/01/1977 possesses R125 (Fig. S5).

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).

Discussion

Our studies show that recent pH1N1 viruses have acquired a significant antigenic mutation that prevents binding of Abs elicited in a large number of middle-aged humans. For this reason, we propose that the pH1N1 vaccine strain should be updated. Conventional serological techniques used by most surveillance laboratories have failed to recognize the K166Q HA mutation as antigenically important (9). HAI assays are based on serial sera dilutions and can only detect large antigenic changes. Many surveillance-based laboratories ignore twofold reductions in HAI titer because these laboratories typically process thousands of samples, which prohibit the experimental precision that is required to reliably detect twofold differences in these assays. However, a true twofold reduction in HAI titer against a mutated strain indicates an extremely immunodominant Ab response. Although we reproducibly detected as low as twofold HAI differences using K166Q HA mutated viruses (Tables S1 and S2 show results from three independent HAI experiments), our HAI assays likely underestimate the number of individuals that possess K166Q Abs. For example, we were able to isolate K166 HA-specific mAbs from pH1N1-vaccinated individuals whose sera yielded similar HAI titers using WT and K166Q mutated pH1N1 viruses. We also identified many human sera samples that had >fourfold reductions in HAI titer using pH1N1 viruses with the K166Q HA mutation, and it is worth noting that these results would likely have been missed if we pooled human sera samples or simply compared overall geometric means of HAI data with mutant viruses.

We attempted to recapitulate K166 HA-specific immunity in ferrets by sequentially infecting with sH1N1 strains and the A/California/07/2009 pH1N1 strain. Only three of eight ferrets sequentially infected with A/Chile/01/1983 and A/California/07/2009 mounted levels of K166 HA-specific Abs that could be detected by HAI assays. Outbred ferrets were used in these experiments, and the overall percentage of ferrets with K166 HA-specificity (Fig. 4) is similar to the overall percentage of humans born in the 1970s with K166 HA-specificity (Fig. 2A). We speculate that variation in K166 HA-specificity in humans is because of variations in pre-exposure histories and genetic differences that impact B-cell repertoires. Studies are ongoing to determine if genetic differences in B-cell repertoires among ferrets influence K166 HA-specificity.

Our results offer a possible antigenic explanation for the increased disease burden in middle-aged adults during the 2013–2014 influenza season. Given that the specificity of Ab responses is altered by pre-exposures, we propose that conventional serological techniques used to identify antigenically novel viruses should be reevaluated. The usefulness of arbitrary HAI titer cutoffs and dependence on antisera generated in previously naïve ferrets (31, 32) should be reconsidered. Although we believe that the pH1N1 vaccine should be updated immediately, it is not clear if a pH1N1 vaccine strain with Q166 HA will be able to break the original antigenic sin that currently exists in some middle-aged individuals. Further studies should be designed to determine if an updated H1N1 vaccine strain with Q166 HA elicits more effective Ab responses in different aged humans with distinct sH1N1 exposure histories.

Materials and Methods

Human Donors.

Studies involving human adults were approved by the Institutional Review Boards of Emory University, Vaccine and Gene Therapy Institute of Florida, the National Institute of Respiratory Diseases of Mexico, and the Wistar Institute. Informed consent was obtained. For all experiments, HAI and in vitro neutralization assays were completed at the Wistar Institute using preexisting and de-identified sera. We analyzed several sera panels in this study. We analyzed sera from healthy donors collected at the New York Blood Center in February of 2014. We analyzed sera from healthy donors collected at the Center for Research in Infectious Diseases at the National Institute of Respiratory Diseases in Mexico. We analyzed sera and mAbs derived from healthy donors vaccinated with a monovalent pH1N1 vaccine in 2009 as previously described (23).

Viruses.

Viruses possessing WT pH1N1 HA or K166Q pH1N1 HA were generated via reverse-genetics using HA and NA genes from A/California/07/2009 and internal genes from A/Puerto Rico/08/1934. All of these viruses were engineered to possess the antigenically neutral D225G HA mutation (15), which facilitates viral growth in fertilized chicken eggs. Viruses were grown in fertilized chicken eggs and the HA genes of each virus stock were sequenced to verify that additional mutations did not arise during propagation. sH1N1 strains (A/USSR/90/1977, A/Chile/01/1983, A/Singapore/06/1986, A/Texas/36/1991, A/New Caledonia/20/1999, and A/Solomon Islands/03/2006) were also grown in fertilized chicken eggs. We isolated a pH1N1 virus from respiratory secretions obtained from a patient from the Children’s Hospital of Philadelphia in 2013 (named A/CHOP/1/13 in this report). For this process, de-identified clinical material from the Children’s Hospital of Philadelphia Clinical Virology Laboratory was added to Madin-Darby canine kidney (MDCK) cells (originally obtained from the National Institutes of Health) in serum-free media with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin, Hepes, and gentamicin. Virus was isolated from the MDCK-infected cells 3 d later. We extracted viral RNA and sequenced the HA gene of A/CHOP/1/13. We also used reverse-genetics to introduce glycosylation sites into A/California/07/2009 (pH1N1) HA. The consensus sequence for N-linked glycosylation (N-x-S/T) was added at HA residues 129, 131, and 163 by making the mutations D131T, D131N and N133T, and K163N, respectively. Similar results were obtained in HAI assays when we used glycosylation mutants grown in eggs or MDCK cells. Glycosylation at residues 129 and 131 was confirmed by treating concentrated virus with PNGase-F (New England Biolabs) under denaturing conditions. The CM1-4 anti-HA1 antibody was used as a primary antibody, and a donkey anti-mouse fluorescent secondary antibody (Licor) was used. Blots were imaged using the Licor Odyssey imaging system at 800nm (secondary antibody) and 700 nm (molecular weight marker).

Animal Experiments.

Murine experiments were performed at the Wistar Institute according to protocols approved by the Wistar Institute Institutional Animal Care and Use Committee. BALB/c mice (Charles River Laboratories) were injected with 25 μg of mAb intraperitoneally and then infected intranasaly with 20,000 TCID₅₀ of WT or K166Q-HA pH1N1 virus 12 h later. As controls, some mice received an intraperitoneal injection of PBS before infection. Weight loss and survival was recorded for 11 d. Severely sick mice were euthanized. Ferret experiments were performed at the Vaccine and Gene Therapy Institute of Florida in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (33), the Animal Welfare Act, and the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories handbook. Fitch ferrets (Marshall Farms) were infected with 1 × 10⁶ PFU of sH1N1 virus and bled 14 and 84 d later. These ferrets were then infected with the A/California/07/2009 pH1N1 strain and bled 14 d later. Some ferrets were sequentially infected with A/California/07/2009 (84 d between infections) and other ferrets were infected with only A/California/07/2009 and bled 14 d later.

HAI Assays.

Sera samples were pretreated with receptor-destroying enzyme (Key Scientific Products or Sigma-Aldrich) and HAI titrations were performed in 96-well round bottom plates (BD). Sera were serially diluted twofold and added to four agglutinating doses of virus in a total volume of 100 μL. Turkey erythrocytes (Lampire) were added [12.5 μL of a 2% (vol/vol) solution]. The erythrocytes were gently mixed with sera and virus and agglutination was read out after incubating for 60 min at room temperature. HAI titers were expressed as the inverse of the highest dilution that inhibited four agglutinating doses of turkey erythrocytes. Each HAI assay was performed independently on three different dates. Sera that had at least twofold reduced HAI titers using K166Q HA mutant viruses in three independent HAI assays were labeled as “K166 HA-specific.”

ELISAs.

Viruses for ELISAs were concentrated by centrifugation at 20,000 RPM for 1 h using a Thermo Scientific Sorvall WX Ultra 80 Centrifuge with a Beckman SW28 rotor. Concentrated viruses were then inactivated by B-Propiolactone (BPL; Sigma Aldrich) treatment. Viruses were incubated with 0.1% BPL and 0.1M Hepes (Cellgro) overnight at 4C followed by a 90-min incubation at 37 °C. The 96-well Immulon 4HBX flat-bottom microtiter plates (Fisher Scientific) were coated with 20 HAU per well BPL-treated virus overnight at 4 °C. Each human mAb was serially diluted in PBS and added to the ELISA plates and allowed to incubate for 2 h at room temperature. As a control, we added the 70-1C04 stalk-specific mAb to verify equal coating of WT and K166Q HA virus. Next, peroxidase conjugated goat anti-human IgG (Jackson Immunoresearch) was incubated for 1 h at room temperature. Finally, Sureblue TMB Peroxidase Substrate (KPL) was added to each well and the reaction was stopped with addition of 250 mM HCl solution. Plates were extensively washed with water between each ELISA step. Affinities were determined by nonlinear regression analysis of curves of 6 mAb dilutions (18 μg/mL to 74 ng/mL) using Graphpad Prism. mAbs were designated as K166-specific if they had a K_d at least four times greater for the K166Q mutant than for the WT virus.

In Vitro Neutralization Assays.

Sera were serially diluted and then added to 100 TCID₅₀ units of virus and incubated at room temperature for 30 min. The virus-sera mixtures were then incubated with MDCK cells for 1 h at 37 °C. Cells were washed and then serum-free media with TPCK-treated trypsin was added. Endpoints were determined visually 3 d later. Data are expressed as the inverse of the highest dilution that caused neutralization. All samples were repeated in quadruplicate and geometric mean titer is reported.

Structural Modeling of HA Glycosylation Sites and Computational and Phylogenetic Analyses of HA Sequences.

Glycans were modeled using the GLYCAM-Web Glycoprotein Builder (www.glycam.org) as described in SI Materials and Methods. Computational and phylogenetic analyses of HA sequences were completed after downloading all full-length human pH1N1 sequences present in the Influenza Virus Resources as of February 23, 2014 as described in SI Materials and Methods.

Statistical Analyses.

For all sera experiments, we excluded samples that did not have positive pH1N1 HAI titers. All samples that were pH1N1 HA-WT HAI-negative were also pH1N1 HA-K166Q HAI-negative. Samples were allocated to specific groups based on age of donor. The year of birth of each sample was available during the experiment, but this information was not assessed until each experiment was completed. Variance of raw HAI titers was similar between different age groups. Fisher’s exact tests were completed using SAS v9.3 software.