Study design and human subjects

Human patients were enrolled as part of an acute influenza surveillance protocol, in which patients presenting influenza-like symptoms were asked to visit the University of Rochester Vaccine Research Unit for sampling by nasal wash and nasopharyngeal swab (combined, nasal wash/swab). Patients were named with a number and the acronym ACU (from Acute influenza). Sera from patients enrolled in the acute influenza surveillance protocol were obtained at the acute visit (day 0), and around 28 days after the acute visit (day 28). Only samples from patients whom tested positive via RT-PCR for IAV H1N1 were included in this study. Some patients had received the 2014–15 or 2015–16 flu vaccine prior to infection (S1 Table). Each study was approved by the University of Rochester Human Research Subjects Review Board (protocol 14–0101). Informed written consent was obtained for each participant.

Cells and viruses

Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293T cells were obtained from the American Type Culture Collection (ATCC, CCL-34 and CRL-11268, rspectively). MDCK cells constitutively expressing the HA protein of A/Indonesia/5/2005 H5N1 (MDCK/H5) were previously described [27]. MDCK and MDCK/H5 cells were grown at 37°C in air enriched with 5% CO₂, in Dulbecco’s modified minimal essential medium (DMEM) (Corning, Manassas, VA; #10-013-CV) supplemented with 10% (v/v) fetal bovine serum (FBS) (Lonza, Walkersville, MD; #14-502F) and 10 μg/mL gentamycin (Gibco, Grand Island, NY; #15710–064). MDCK/H5 cells were grown in media additionally supplemented with 200 μg/mL hygromycin B every other passage to ensure continued expression of H5, as previously described [27,28].

A virus stock of influenza virus A/California/04/2009 (A/Cal/04/09), produced in 9 to 11-day-old SPF embryonated chicken eggs, incubated 2 to 3 days at 33.5 C, was used as the reference strain for HAI and MN assays (BEI Resources, Manassas, VA; NR-13659).

Virus isolation and titrations

MDCK cells were inoculated with nasal washes/swabs from pH1N1-like virus-infected patients. Upon observation of cytopathic effect, supernatants were collected and used to infect fresh cells. If no cytopathic effect was observed, supernatants were collected 72 hours post-infection (hpi) and used to infect fresh cells. A maximum of three serial passages was performed. All infected cells were grown at 37°C in air enriched with 5% CO₂, in DMEM supplemented with 0.3% bovine serum albumin (BSA) (Gibco; #15260–037), 10 μg/mL gentamycin, and 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemical, Lakewood, NJ; #3570).

Viruses were titrated by immunofocus assay using an IAV monoclonal Ab specific for the viral NP protein (HB-65), as previously described [29,30]. IAV NP-positive cells were visualized and enumerated to determine virus titers (fluorescent-forming units, FFU/ml) using a fluorescence microscope.

HA and NA sequencing

Viral RNA was purified from nasal swabs/washes using the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) or from cell culture extracts using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed to obtain cDNA of the HA or NA genes using primers H1N1-HA-5’NCR-VS (5’-GGGGAAAACAAAAGCAACAAAAATG-3’) and H1N1-HA-3’-NCR-RS (GTGTTTTTCTCATGCTTCTGAAATCCTAATG-3’) or NA-A/Cal-1-VS (5’-ATGAATCCAAACCAAAAGATAATAACCATT-3’) and NA-A/Cal-1410-RS (5’-TTACTTGTCAATGGTAAATGGCAACTC-3’), respectively, with the High Capacity cDNA reverse transcription kit (Applied Biosystems; #4368814).

To obtain DNA segments encoding the HA or NA genes to sequence the HA and NA genes, overlapping PCR using primers H1N1-HA-5’NCR-VS and H1N1-HA-923-RS (GGGAGGCTGGTGTTTATAGCACCC-3’) (for HA region 1) and H1N1-HA-793-VS (CTAGTGGTACCGAGATATGCATTCGC-3’) and H1N1-HA-3’-NCR-RS (for HA region 2) or NA-A/Cal-1-VS and NA-A/Cal-755-RS (5’-GAGGCCTGTCCATTACTTGGTCC-3’) (for NA region 1), and NA-A/Cal-652-VS (5’-GAGTTGGAGAAACAATATATTGAGAACACAAG-3’) and NA-A/Cal-1410-RS (for NA region 2), respectively, was performed with the Platinum Pfx polymerase (Life Technologies; #11708021). The VS and RS primers used in each PCR were used for Sanger sequencing. The complete HA and NA sequences were obtained directly amplifying the viral RNA from nine of the nasal swabs/washes. In addition, viruses from 9 of the 14 clinical samples were isolated, and the complete HA (GenBank: KY056604, KY056601, KY056612, KY056603, KY056600, KY056608, KY056614, KY056613, KY056606) and NA (GenBank: KY056597, KY056599, KY056602, KY056609, KY056598, KY056607, KY056605, KY056610, KY056611) sequences were obtained from the viruses isolated in MDCK cells.

Plasmids

Ambisense pDZ plasmids [31] encoding the NA genes of A/Cal/04/09 (NA-wt) or the virus isolated from patient 001 (NA-001) were obtained by overlapping PCR. Viral RNA purified from the nasal swab/wash purified from patient 001 (NA-001) or cells infected with A/Cal/04/09 (BEI Resources, NR-13659) (NA-wt) was reverse transcribed using primers NCR-SapI-NACal-5-VS (5’-GATCGCTCTTCTGGGAGCAAAAGCAGGAGTTTAAAATGAATCCAAACCAAAAG-3’) and NCR-SapI-NACal-3-RS (5’-CATCGCTCTTCTATTAGTAGAAACAAGGAGTTTTTTGAACAAATTACTTGTCAATGGT-3’) and the High Capacity cDNA reverse transcription kit (Applied Biosystems) to produce cDNA. These cDNAs were then amplified by PCR using the same primers NCR-SapI-NACal-5-VS and NCR-SapI-NACal-3-RS and the Platinum Pfx polymerase (Life Technologies) to obtain the DNA transcript of the NA gene with SapI restriction sites at the 5’ and 3’ ends. PCR products were digested with the SapI restriction enzyme and cloned into the pDZ plasmid.

Virus rescues

H5-pseudotyped single cycle infectious IAVs (sciIAVs) in which the viral HA gene was replaced by a gene encoding green fluorescent protein (GFP) and the viral NA gene was replaced either by the NA encoded by A/Cal/04/09 (NA-wt) or by the virus isolated from patient 001 (NA-001) were generated in the backbone of A/Cal/04/09 as previously described [27,32]. Briefly 293T cells and MDCK cells stably expressing the HA protein from the H5N1 strain A/Indonesia/5/2005 (MDCK/H5) [27], complementing the absence of the HA gene in the sciIAV genome, were co-cultured at a 1:1 ratio in 6-well plates were transiently co-transfected, in suspension, with 1 μg each of ambisense plasmids encoding the six A/Cal/04/09 genes (excluding HA and NA) (pDZ-PB2, -PB1, -PA, -NP, -NS, -M, and -GFP) (kindly provided by A. Garcia-Sastre, Icahn School of Medicine at Mount Sinai, NY) and GFP plus the pDZ-NAs encoding NA-wt or NA-001 proteins, using DNA-IN (Molecular Transfec, Inc; #73771) [27,28]. Stocks were titrated by immunofocus assay (FFU/ml) on MDCK cells as described above. The identity of the NA ORFs in the rescued viruses was confirmed by restriction digestion and sequencing (Genewiz). Before using the sciIAVs in assays, it was confirmed that neither the ferret nor human sera samples contained antibodies with HAI activity against these H5-pseudotyped viruses to avoid potential interference that could influence measurements of NAI Ab titers.

Hemagglutination inhibition (HAI) and microneutralization (MN) assays

Sera from ferrets infected with A/Cal/07/09 (obtained from BEI Resources; NR-15429) and patient sera collected at days 0 and 28 were tested in duplicate against A/Cal/04/09 and the virus isolated from patient 022 for hemagglutination-inhibiting (HAI) and HA-specific neutralizing Ab titers.

For HAI assays, twofold serial dilutions of human or ferret sera treated with receptor destroying enzyme (RDE) (Denka Seiken, Campbell, CA) were mixed with 8 HA units/50 μL of each virus. The HA protein of the A/Cal/04/09 virus used in the assays was identical to the one deposited in GenBank (accession number JF915184.1). The serum/virus mixture was incubated with 0.5% turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA) for approximately 30 minutes at room temperature. The HAI Ab titer was measured as the highest dilution of serum at which hemagglutination was inhibited.

For MN assays, twofold serial dilutions of human or ferret sera was mixed with approximately 200 FFU of each virus and was left at room temperature for 60 minutes. Confluent MDCK cell monolayers were then inoculated with the serum/virus mixtures. After an adsorption period of 60 minutes at room temperature, serum/virus mixtures were removed and replaced with DMEM supplemented with 0.3% BSA, 10 μg/mL gentamycin, and 1 μg/mL TPCK-trypsin. After incubation for 8 hpi at 37°C in air enriched with 5% CO₂, infected cells were fixed and permeabilized with 0.5% Triton X-100 and 4% formaldehyde in PBS, and an immunofocus assay was performed to determine the amount of not-neutralized virus. The HA-specific neutralizing Ab titer was measured as the highest dilution of serum at which more than 50% of FFU were inhibited.

Recombinant influenza HA proteins

All recombinant (r)HA proteins, encoding a C-terminal trimerization domain and a hexahistidine purification tag [33] were cloned in the plasmid pFastBac CT-TOPO vector (Invitrogen, Grand Island, NY) and expressed using a baculovirus system, as previously described [34]. Purified rHAs were concentrated and desalted with 30 kDa Amicon Ultracell centrifugation units (Millipore, Billerica, MA) and re-suspended in PBS. The purity, integrity and identity of rHA proteins was assessed by NuPage 4–12% Bis-Tris gels (Invitrogen, Grand Island, NY) and Western blot. Western blot analysis was performed using polyclonal rabbit anti-influenza strain/subtype specific primary (eEnzyme, Gaithersburg, MD) and goat anti-rabbit horseradish peroxidase conjugated secondary (Bio-Rad, Hercules, CA) antibodies. Protein concentration was quantified using the Quickstart Bradford Dye Reagent (Bio-Rad, Hercules, CA) with a bovine serum albumin standard curve.

Purified rHA proteins for each influenza strain/subtype were covalently coupled to Bio-plex Pro™ Magnetic COOH Beads (Bio-Rad, Hercules, CA) using the Bio-Plex Amine Coupling Kit (Bio-Rad, Hercules, CA) as previously described [34]. The coupled beads were tested with anti-HA subtype specific ferret or rabbit polyclonal antibodies (Influenza Reagent Resource, IRR, Manassas, VA) and a goat anti-rabbit or anti-ferret phycoerythrin (PE)-conjugated detection Ab (SouthernBiotech, Birmingham, AL).

mPlex-Flu assay

Quantitation of Ab levels in human sera was performed as previously described [34]. Briefly, a panel of HA-coupled mPlex-Flu beads were mixed and incubated with diluted human serum, at 500 beads per each HA to be detected, in 96-well filtration plates (Millipore, Billerica, MA) at 4°C overnight, on a rotary shaker (500 rpm), in the dark. Wells were washed twice and then incubated with PE-conjugated anti-mouse IgG (γ chain specific, at 1:400 dilution) secondary antibodies (SouthernBiotech, AL) in the dark at room temperature for 2 h with gentle agitation (500 rpm). After 3 additional washes, the beads in each well were resuspended in Luminex Magpix Drive Fluid (Luminex, Austin, TX), analyzed on a Magpix multiplex reader (Luminex, Austin, TX). The results were expressed as median fluorescence intensity (MFI). The infected subject’s sera were diluted 1: 5,000 and 1:10,000 and the absolute concentration of anti-HA antibodies present in serum were calculated from a standard curve which was generated at the same time as previously described [34]. Average concentrations of each anti-HA antibody from both serum dilutions was shown in the heatmaps.

Enzyme-linked lectin assay (ELLA)

Sera from ferrets infected with A/Cal/07/09 (BEI Resources; NR-15429) and patient sera collected at days 0 and 28 were tested in duplicate against sciIAV-NA-wt and sciIAV-NA-001 for NAI Ab titers using a modified enzyme-linked lectin-based assay (ELLA), as previously described [19,35,36]. Bound lectin was detected with 3,3',5,5'-tetramethylbenzidine (TMB) (Thermo Scientific #34028), and optical density was measured at 450 nm. Each virus was titrated by ELLA using 10-fold serial dilutions in the absence of serum, and the dilution for each virus at which the optical density at 450 nm was closest to the maximum signal but within the linear range of the titration curve was selected to maximize assay sensitivity and precision.

At least 8 wells on each plate were dedicated to positive (no serum; virus only) or negative (blank; no serum or virus) controls. The average background signal (negative control) was subtracted from all readings, and percent inhibition compared to the average maximum signal (positive control) at each serum dilution was calculated. The NAI Ab titer was measured as the highest dilution at which at least 50% of the maximum signal was inhibited. Only assays in which the background values were less than 10% of the maximum signal were considered.

Publically available HA and NA sequences

19,926 HA and 17,430 NA DNA sequences of pH1N1-like strains isolated from humans since 2009 were downloaded from the GISAID online database at the time of investigation (http://platform.gisaid.org/epi3/frontend). 16,981 HA and 15,796 NA DNA sequences remained after removing sequences lacking associated metadata, or containing missing or erroneous (not A, C, T, or G) nucleotides. The HA (9) and NA (9) sequences from the Rochester, NY viral isolates were added to the publically available sequences, resulting in 16,990 HA and 15,805 NA DNA sequences which were used for all sequence-based analyses. Sequences were aligned using MUSCLE [37,38].

Phylogenetic reconstructions

Phylogenies were reconstructed with the neighbor-joining method and bootstrapped 1000 times with ClustalX version 2.1, using 16,990 HA or 15,805 NA aligned DNA sequences [39]. Each individual branch of the phylogenetic trees were colored according to the season during which the strain encoding each HA or NA protein was isolated. Phylogenetic trees were rendered in FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Clades were identified as previously described [8].

Selection analyses

A single-likelihood ancestor counting (SLAC) approach [40] was used to infer the non-synonymous (dN) and synonymous (dS) substitution rates on a per-site basis under the HKY85 evolutionary model for 16,990 HA or 15,805 NA aligned DNA sequences encoded by pH1N1-like strains and phylogenies reconstructed using ClustalX (see above) with HyPhy MPI [41]

Sequence-based mapping

For each HA sequence, we calculated its hamming distance to all other HA sequences in the data set, as previously described [42]. The hamming distance is the number of amino acid differences at each residue’s position in the hemagglutinin protein. This process results in a distance matrix consisting of the number of amino acid differences between all viruses in the data set. Classical multidimensional scaling (principal coordinates analysis) was performed on the distance matrix using the stats package in R.

Statistical analyses

Log2 transformation was applied to Ab titers to stabilize data variability and normalization. Repeated ANOVA was used to simultaneously study multiple effects (string and time), and to take account of within-subject correlations. Multiple testing adjustment of Tukey method was applied to prevent type I error inflation. The findings were confirmed by pairwise comparisons through Sign test.

The HA and NA proteins encoded by 2015–2016 influenza virus isolates from Rochester, NY encode amino acid differences compared to the strain used for vaccination that same season

Influenza virus HA and NA proteins undergo antigenic drift, leading to decreased vaccine efficacy [21,43,44]. Previously, it was shown that risk of infection with influenza during the 2010–2011 and 2012–2013 flu seasons may have been due to a poor response to vaccination as well as contact with antigenically distinct IAV strains [26]. To analyze genetic and antigenic variation within the HA and NA proteins of pH1N1-like viruses circulating during the 2015–2016 season, 14 nasal swabs/washes were collected in Rochester, NY. All mutations described are numbered by their position along the amino acid sequence relative to the methionine codon at the beginning of the N-terminal signal peptide (position 1). All comparisons, both sequence-based and serological assays, were performed using A/Cal/04/09, another early isolate, as the reference strain [45]. H1N1 viruses emerging in 2009 were antigenically homogenous [46]. The HA protein sequence of A/Cal/04/09 differs from that of A/Cal/07/09 by 2 non-synonymous mutations at positions 200 and 214, and the NA protein sequences of the two strains are identical (see Tables 1 and 2, respectively). With these differences taken into consideration, A/Cal/04/09 was used to represent the vaccine strain throughout this paper.

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Table 1. Mutations present in the HA protein of 2015–2016 circulating pH1N1-like strains compared to the vaccine strain.

https://doi.org/10.1371/journal.pone.0188267.t001

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Table 2. Mutations present in the NA protein of 2015–2016 circulating pH1N1-like strains compared to the vaccine strain.

https://doi.org/10.1371/journal.pone.0188267.t002

Influenza viruses present in nasal washes/swabs from subjects infected during the 2015/2016 season encoded 18 amino acid changes in the HA protein compared to the strain Cal/04/2009, circulating at the origin of the 2009 pandemic (Table 1). The infecting viruses were isolated in MDCK cells from 9 out of the 14 nasal swabs/washes and HA sequences were obtained from the viruses (Table 1). The HA sequences of viruses grown in MDCK cells were the same as those of viruses present in the original nasal washes/swabs (data not shown), with the exception of viruses isolated from patients ACU007 and ACU017, that introduced an additional mutation S207R (Table 1). As with the majority of H1N1 viruses circulating during the 2015–2016 season, these isolates were classified as clade 6B.1 [8,25].

Out of these 18 mutations, 7 were located in the HA stalk domain while the other 11 were located in the HA head domain (Table 1). As shown on a 3D model of the HA protein (RCSB PDB ID: 3LZG) [47], 5 mutations were found in previously characterized [4] Ab-binding sites: S179N and K180Q, in Sa; P200S and S202T, in Sb; and S220T, in Ca1 (Fig 1A and Table 1) [9]. Three of the mutations (P200S and S202T close to the 190 alpha helix, and I233T along the 220 loop) were in close proximity to the receptor-binding domain (Fig 1B) [3,5,48]. The 190 helix and the 220 loop are numbered with respect to the structure-based sequence alignment of different HA subtypes based on H3 numbering [49]. All isolated viruses encoded the same HA head domain except for virus ACU009, which lacked the S179N mutation. Additionally, there were variations in the HA stalk domain between the viruses infecting the 9 patients, as only two viruses (infecting patients ACU004 and 017) encoded substitution T472N (Table 1).

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Fig 1. Mutations present in the HA or NA proteins of 2015–2016 circulating pH1N1-like strains compared to the vaccine strain.

Three-dimensional models of the A/Cal/04/09 HA (A and B) or NA (C and D) proteins (PDB ID: 3LZG and 3NSS, respectively), showing the mutations of circulating pH1N1-like viruses compared to that of the vaccine strain. A top and views of the HA or NA protein quaternary structures are shown. The HA antigenic sites Sa (dark blue), Sb (yellow), Ca1 (light purple), Ca2 (dark purple), and Cb (light blue) are indicated. The HA fusion domain is represented in orange [52,53]. A bracket (not to scale) indicates the location of the NA stalk had it been resolved in the crystal structure. Known antigenic sites on the NA protein are represented in yellow. The mutations present in the HA or NA of pH1N1-like circulating strains compared to that of the H1N1 vaccine strain are shown in red. The approximate location of HA mutation S220T, situated in the Ca1 antigenic site at the subunit interface, is indicated. The ellipse indicates the receptor-binding site (RBS) of the HA (B) or the catalytic site of the NA (D), shown at a higher magnification. 3D model was customized using PyMOL.

https://doi.org/10.1371/journal.pone.0188267.g001

As for the NA protein (sequenced from nasal swabs/washes, as well as the 9 infecting viruses isolated in MDCK cells), 14 mutations were found when compared to the vaccine strain (Table 2). As seen on a 3D model of the NA protein (RCSB PDB ID: 3NSS) [50], 5 out of the 14 mutations (N200S, N248D, I321V, N369K, and K432E) were at previously characterized Ab-binding sites (Fig 1C) [51]. Three of the mutations (K432E along the 430 loop, V241I and N248D) were located near the catalytic site (Fig 1D). The 430 loop is numbered with respect to structure-based sequence alignments based on N2 numbering [50]. All isolates encoded the same NA sequence, with the exception of viruses infecting patients ACU008 and 022, which did not encode the mutation I34V. Altogether, these data indicate genetic variability in both, the HA and NA proteins of currently circulating H1N1 viruses, compared to the vaccine strain.

The HA and NA proteins encoded by influenza virus isolated in Rochester (NY) are representative of the HA and NA proteins from viruses circulating worldwide during the 2015–2016 season

To analyze if the HA and NA protein sequences of viruses isolated in the Rochester, NY area, were representative of viruses circulating globally, these HA and NA sequences were compared to those of pH1N1-like viruses isolated worldwide during the 2015–2016 flu season. Out of the 6,552 HA and 5,462 NA sequences of 2015–2016 isolates uploaded to the GISAID database at the time of investigation, the majority of isolates from different geographic regions encoded HA and NA proteins with mutations identical to those described here (S2 and S3 Tables, respectively). These data indicate that the HA and NA proteins encoded by viruses isolated in Rochester, NY (Tables 1 and 2, respectively), are representative of those encoded by the dominant pH1N1-like strains circulating during the 2015–2016 season in all geographic regions (S2 and S3 Tables, respectively).

pH1N1-like HA and NA glycoproteins show directional selection of mutations in antigenic sties

To analyze selection pressures at the sites of mutations present in circulating strains, selection analyses (dN/dS ratio) [54–56] were performed on 16,990 HA or 15,805 NA DNA sequences of pH1N1-like strains isolated from humans since 2009. The dN/dS ratio is the ratio of the rate of non-synonymous mutations (dN) to the rate of synonymous mutations (dS) at each codon. As such, dN/dS has been used as a measure of evolutionary change by giving an estimate of the magnitude and direction (positive or negative) of selective pressure acting on a codon [41,55,57,58]. Of the 566 amino acids comprising the HA protein of pH1N1-like strains, 12.7% (72 of 566) of the codons were subject to positive (diversifying) selection (dN/dS > 1), while 87.3% of codons (494 of 566) were under negative (purifying) selective pressure (dN/dS < 1) (Fig 2A and 2C) [41,55]. There were more codons under positive selective pressure in the HA head region (19.7%) than the HA stalk region (7.8%). Interestingly, 12 of the 18 amino acids (positions 13, 100, 101, 114, 180, 200, 202, 214, 220, 273, 391, and 472), at which mutations were found in the HA of 2015–2016 isolates, were under positive selective pressure (Fig 2E), suggesting that these mutations are the result of positive (diversifying) selection. Four of these amino acids are located in antigenic sites in the HA.

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Fig 2. dN/dS selection analysis for pH1N1-like HA or NA proteins.

A selection analysis (dN/dS ratio) was performed on 16,990 HA (A and C) or 15,805 NA (B and D) DNA sequences of pH1N1-like strains isolated from humans since 2009, and the dN/dS ratio at each codon along the HA (A) or NA (B) DNA sequence was plotted. The dN/dS ratio for codons at which mutations were found in 2015–2016 isolates are shown in green. The lower bar represents amino acid residues along the HA or NA proteins corresponding to the head domain (gray), the stalk domain (black), the transmembrane domain (TMD) (red), and the cytoplasmic tail (CT) (blue). Three-dimensional model of the HA (PDB ID: 3LZG) (C) or the NA (PDB ID: 3NSS) (D) proteins, with amino acid residues colored according to the dN/dS ratio at the corresponding codon using PyMOL. A bracket (not to scale) indicates the location of the NA stalk had it been resolved in the crystal structure. (E and F) Tables showing the dN/dS ratio for the codons at which amino acid substitutions were present in the HA (E) or NA (F) of pH1N1-like viruses circulating during 2015–2016. Gray shadows indicate codons at positions within known Ab-binding sites.

https://doi.org/10.1371/journal.pone.0188267.g002

Of the 469 amino acids in the NA protein of pH1N1-like strains, 11.3% (53 of 469) of codons were subject to positive selection, while 88.7% of codons (416 of 469) were under negative selective pressure (Fig 2B and 2D). Interestingly, seven of the 14 amino acid residues (positions 34, 40, 200, 248, 270, 321, and 386) at which mutations were found in the NA of 2015–16 isolates, three of which are located in antigenic sites, were under positive selective pressure (Fig 2F).

As expected, mutations have been incorporated by the pH1N1-like virus population over the past seven seasons, beginning with the emergence of the pandemic-causing triple reassortant in early 2009 [8,59,60]. Notably, strains encoding an HA protein with five of the mutations (P100S, P200S, S220T, I338V, and T214A) became the dominant circulating strains early in 2009 following detection of the outbreak (Fig 3). With respect to the NA protein, only one mutation, N248D, appeared in the NA protein of circulating strains immediately following the outbreak in early 2009 (Fig 4). The remaining mutations in the HA and NA proteins were introduced in strains circulating during following seasons (Figs 3 and 4). Importantly, once each mutation was introduced into the HA (Fig 3) or NA (Fig 4) by pH1N1-like strains, the percentage of total pH1N1-like isolates encoding the mutation increased each season and remained prevalent in isolates of following seasons. The order in which mutations were introduced corresponds with the phylogenetic analysis of the HA (Fig 5A) or the NA (Fig 5B) of pH1N1-like strains performed by us as well as others [8,59,60]. These data suggest evolution and selection pressure on the HA and NA proteins from pH1N1 viruses circulating since 2009.

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Fig 3. Frequency of HA amino acid changes found in pH1N1-like viruses over time.

Publicly available sequences of the HA protein of pH1N1-like strains isolated since 2009 (n = 16,990) were analyzed, and were plotted according to the percentage of sequences containing the original (incumbent) amino acid present in the H1N1 vaccine strain (white) or the substitute amino acid (black) in pH1N1-like isolates at positions 100, 200, 338, 214, 220, 391, 114, 468, 202, 516, 300, 273, 180, 13, 101, 179, 233, 472 from each season since early 2009. An (*) next to the amino acid location indicates that the position is in a previously described antigenic site.

https://doi.org/10.1371/journal.pone.0188267.g003

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Fig 4. Frequency of NA amino acid changes found in pH1N1-like viruses over time.

Publicly available sequences of the NA protein of pH1N1-like strains isolated since 2009 (n = 15,805) were analyzed, and were plotted according to the percentage of sequences containing the original (incumbent) amino acid present in the H1N1 vaccine strain (white) or the substitute amino acid (black) in pH1N1-like isolates at positions 13, 34, 40, 44, 200, 241, 248, 264, 270, 314, 321, 369, 386, and 432 from each season since early 2009. An (*) next to the amino acid location indicates that the position is in a previously described antigenic site.

https://doi.org/10.1371/journal.pone.0188267.g004

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Fig 5. The genetic relatedness of the HA and NA proteins encoded by pH1N1-like viruses circulating since 2009.

Phylogenetic reconstruction of HA (A, n = 16,990) and NA (B, n = 15,805) proteins encoded by pH1N1-like viruses. Clades are indicated to the left; branches are colored according to season of isolation.

https://doi.org/10.1371/journal.pone.0188267.g005

The mutations in the HA protein encoded by circulating viruses contribute to the evasion of HA-binding antibodies

HA is the main viral protein inducing antibodies that protect against infection [61]. To analyze the relevance of the mutations in the HA protein in evading serum antibodies induced by previous exposure/vaccination, HAI and MN assays were performed using serum from ferrets raised against A/Cal/07/09 and human sera obtained from 14 patients during the acute visit (approximately 2–3 days after infection; day 0) and the post-acute visit (28 days after the acute visit; day 28, not available for patients 001, 007, 017, 023, and 024) (Fig 6). Virus isolated from patient 022 was used and referred to as the circulating strain, as it comprises the mutations common to the HA of pH1N1-like strains isolated worldwide during the 2015–2016 flu season (S2 Table and Table 1). Given that sera was obtained from patients who were infected, it was not surprising that HAI Ab titers were low (< 40, in 9 out of 14 patients) in human sera collected at day 0, even in patients who were vaccinated that season (patients 003, 004, 008, 009, 010, 017, 022, 023, and 024), and only one subject (010) showed a titer >40 (Fig 6A). These data showing low HAI titers even in vaccinated subjects suggests poor responses to the pH1N1 component of the vaccine. Globally, at day 28, HAI Ab titers significantly (P<0.005) increased in patient serum, as expected [26], with the exception of patient 009. At day 0, HAI Ab titers using human sera from patients 005, 008, and 012 were similar for both circulating and vaccine strains (Fig 6A). However, HAI Ab titers in the sera of patients 004, 007, 010, 017, and 024 at day 0, and in serum from ferrets infected with A/Cal/07/09 virus (Fig 6A and 6B) were demonstrably lower (at least 2-fold) for the circulating strain than for the vaccine strain, showing that the 18 mutations found in the viruses which circulated during the 2015–2016 season affected HA protein antigenicity to a limited degree.

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Fig 6. Effect of mutations present in the HA ofin 2015–2016 isolates on the antigenicity of the viral protein.

HAI (A and B) and MN (C and D) assays using human (A and C) and ferret (B and D) antisera. Standardized antisera from ferrets infected with the vaccine virus, and human sera collected from infected subjects at the acute (day 0) and post-acute visits 28 days later (day 28), were measured using HAI or MN assays for antibodies specific for the H1N1 vaccine strain (VV) and the virus isolated from patient 022 (IV). The HA protein of the virus isolated from patient 022 encodes mutations found in the majority of 2015–2016 isolates circulating worldwide (S2 Table and Table 1). Individual subject numbers from which the viruses were isolated are shown in the legend. Subjects whose titers are represented with circles received the 2015–2016 flu vaccine, while those represented with triangles were not vaccinated (see S1 Table). Experiments were repeated three times, showing reproducible data. *, indicates p-values <0.05 using a Student’s t-test in B and D. IV, isolated virus. VV, vaccine virus. HAI Ab titer of 40, commonly associated with protection, is indicated by an arrow in (A) and (B) [62,63]. The dotted line indicates the limit of detection (LoD), at an Ab titer of 10. A titer of 5 was considered for titers that fell below the (LoD).

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In MN assays, serum Ab titers specific for the infecting and vaccine strains were significantly (P<0.005) higher after infection (day 28), as expected. The only exception was the titers specific for the vaccine strain, which did not increase in patients 009 and 010 (Fig 6C). Interestingly, at day 0, HA-specific neutralizing Ab titers in serum from patients 003, 004, 005, 007, 009, 018, and 022 were 2-fold lower for the circulating strain than for the vaccine strain, and neutralizing Ab titers in the serum of patients 010, 017, and 024 were 4-fold lower for the circulating strain than for the vaccine strain (Fig 6C). Using ferret serum, we observed over 2-fold difference in neutralizing Ab titers specific to the vaccine strain compared to the circulating strain (Fig 6D). Taken together, the data from the HAI assays and the MN assays indicate that at least one of the mutations found in the HA of circulating viruses make a measurable but limited contribution to the evasion of HA-Ab binding.

The Ab response after infection is adapted to the HA of the pH1N1-like infecting virus

Circulating levels of HA-reactive Ab levels in humans are often highest against HA proteins related to those of viruses that caused significant early-life influenza virus exposure. Generally, later infections/vaccinations maintain these patterns long-term by preferential boosting of cross-reactive anti-HA Ab levels, a phenomenon known as “original antigenic sin (OAS)” (reviewed in [64]). To relate the Ab response to the HA of the infecting virus to OAS-type production of H1-reactive Abs, we used a multiplex assay to measure the levels of Abs reactive to a range of H1 variants (Fig 7). Binding was also measured against a range of H3 variants, other influenza A HA subtypes, influenza B HAs, and chimeric proteins (cH5/1 and cH9/1). The chimeric proteins, encoding “exotic” H5 or H9 head domains and a A/Cal/04/09 HA stalk domain were included to preferentially measure the induction of antibodies specific for the HA stalk domain after infection. A sequence-based analysis was performed to visualize the amino acid variability among the different HA subtypes, and among the different H1 strains, as previously described [42] (S1A and S1B Fig). Importantly, the HA protein from A/Michigan/45/2015 H1N1 strain encoded the same amino acid sequence as the virus isolated from patient 022, used for the HAI and MN assays. All the sera from pH1N1-like virus-infected subjects extracted at day 0 (acute visit) showed low binding to A/Cal/04/09 and A/Michigan/45/2015 HA proteins (Fig 7A), being the sera from patient 010 the one showing the highest (but still low) binding (Fig 7A). These results are consistent with the low HAI titers detected in HAI assays, in which the highest titer at day 0 (80) was observed for this same patient 010 (Fig 6A). Antibodies specific to the HA proteins from A/Cal/04/09 and A/Michigan/45/2015 increased from 1.9 to 10-fold in all the subjects at day 28, compared to day 0, with the exception of patient 009 (Fig 7A and 7B), as observed in the HAI assays (Fig 6A). In addition, whereas at day 0 subject 010 sera bound A/Cal/04/09 HA protein to a higher degree than A/Michigan/45/2015 (as shown in Fig 6A for the HAI titers), at day 28, human sera from subjects 004, 005, 012, 018, and 022 showed a slightly higher reactivity to the HA protein from A/Michigan/45/2010 strain than to the HA protein from A/Cal/04/09 strain (Fig 7A). These data are consistent with an immune response adaptation to the infecting virus, and indicated that the HA proteins from A/Cal/04/09 and A/Michigan/45/2015 (encoding the same amino acid sequence to the HA protein from most 2015/2016 circulating strains) are not antigenically identical since different human sera do not bind both of them to the same extent.

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Fig 7. Binding of serum from pH1N1-infected subjects to HA proteins from different strains.

(A) Concentrations of sera IgG antibodies cross-reacting to each HA protein were measured by multiplex assay against the indicated IAV and IBV HA proteins. Heatmap showing the average Ab concentrations using two human sera dilutions (1:5,000 and 1:10,000) in duplicates. Subject number, subject year of birth and visit of sera extraction (d0 or d28) are indicated on the left. (B) Graphs representing the fold-change in binding to HA proteins using the sera from day 0, compared to the sera from day 28. Each graph represents one subject. For the HA protein strains: Blue colors represent H1N1 strains (A/South Carolina/1/1918, SC18; A/Puerto Rico/8/1934, PR8; A/USSR/90/1977, USSR77; A/Texas/36/1991, Tex91; A/New Caledonia/20/1999, NewCal99; A/California/04/2009, Cal09; A/Michigan/45/2015, Mic15), black color represents an H2N2 strain (A/Japan/305/1957, Jap57), pink colors represent H3N2 strains (A/HongKong/1/1968, HK68; A/Port Chalmers/1/1973, PC73; A/Alabama/1/1981, Ala81; A/Philippines/2/1982, Phi82; A/Panama/7/1999, Pan99; A/Wyoming/2003, Wyo03; A/Hiroshima/52/2005, Hir05; A/Wisconsin/67/2005, Wis05; A/Perth/16/2009, Perth09; A/Victoria/361/2011, Vic11, A/Texas50/2012, Tex12, A/Switzerland/2013, Swit/2013), blue color represents H5N1 (A/Vietnam/1204/2004, Viet04), purple color represents H6N1 (A/Taiwan/2/2013, TW13), fucsia colors represent H7N9 (A/Shanghai/2/2013, SH13 and H7N1 (A/rhea/North Carolina/39842/93, rhea/NC93), green color represent B (B/Brisbane/60//2008, Bris08; B/Phuket/2013, Phu13), and gray colors represent H5, and H9 head, and chimeric proteins cH5/1 and cH9/1.

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Serum Ab binding to the chimeric HAs cH5/1 and cH9/1 increased more than 1.5-fold in 8 of 9 subjects, indicating induction of Abs reactive to the stalk region of H1 (a group 1 HA) (Fig 7A and 7B). In most of the subjects, there was an slight increase in Ab binding to other group 1 HAs (H2, H5, and H6), consistent with production of Abs specific for the conserved stalk (Fig 7A and 7B), as it has been shown in other studies demonstrating an anti-HA stalk Ab response following influenza infection in humans [17,65,66]. Ab binding to the group 2 HAs H3 and H7 changed very little or nothing from days 0 to 28 (Fig 7A and 7B), indicating that pH1N1 infection generates little anti-stalk Ab that cross-reacts between HA groups, and likely reflecting that the HA stalk domain differs significantly between the two groups of HA proteins [67].

Three subjects had relatively high day 0 Ab levels against H1s of older H1N1 viruses that were consistent with OAS-type patterns: subjects 010 (b. 1963) and 018 (b. 1973) had high day 0 titers against H1 USSR77, and subject 012 (b. 1991) had high titers against H1 Tex91 (Fig 7A). In most subjects, H1N1 infection (day 28 sera) increased day 0 Ab levels against older H1s and maintained the day 0 binding profiles, consistent with production of Abs that cross-reacted with H1 variants. The magnitude of these increases was not accounted for by increased levels of stalk-specific Abs (Fig 7B), indicating that the cross-reactive response was directed at HA head epitopes. Notably, the most marked increase in HA-binding Ab using sera collected at day 28 was against the H1s of A/Cal/04/09 and A/Michigan/45/2015 (Fig 7B), indicating that OAS-type responses were not at the expense of Ab adaptation to the infecting virus. OAS-type patterns of H3-binding Abs on day 0 were evident in most subjects, but these levels remained largely unchanged after H1N1 infection. For example, using sera from day 0, we found that patient 010 (b. 1963) preferentially recognized proteins from H3N2 viruses circulating in 1968 (HK68), 1973 (PC73) and 1982 (Phi82). Patients 018, 009 and 022 (b. 1973, 1976, and 1981 respectively) preferentially recognized the H3 proteins from 1981 and 1982 strains. Patients 001 and 005 (b.1989 and 1992, respectively) recognized best the H3 protein from A/Panama/99 than the H3 proteins circulating before 1992 and after 1999, likely reflecting a first exposure to a similar A/Pan99 strain (Fig 7A).

Mutations in the NA protein encoded by circulating viruses contribute to the evasion of NA-binding antibodies

Vaccination and infection induce NA-specific protective antibodies [16,20]. It has been shown that antibodies in complex with HA sterically interfere with NA–Ab binding [68,69]. Therefore, in order to analyze NAI Ab titers, we performed enzyme-linked lectin assays (ELLAs) with H5-pseudotyped sciIAVs expressing the NA protein encoded by A/Cal/04/09 or by the circulating strain isolated from patient 001, which comprised the mutations common to the NA of pH1N1-like strains isolated worldwide during the 2015–2016 flu season, and the HA protein from a H5 virus (A/Indonesia/5/2005). Vaccine strain- and circulating strain-specific NAI Ab titers were measured using patient sera collected at day 0 and 28 days later, as well as serum from ferrets infected with A/Cal/07/09 virus (Fig 8).

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Fig 8. Effect of mutations present in the NA of 2015–2016 isolates on the antigenicity of the protein.

ELLA assays using human (A) and ferret antisera (B). Standardized antisera from ferrets infected with the vaccine virus, and human sera collected from infected subjects at the acute (day 0) and post-acute visits 28 days later (day 28), were measured using ELLA for NAI antibodies specific for the NA protein of the vaccine strain and the virus isolated from patient 001, which encodes mutations found in the majority of 2015–2016 isolates circulating worldwide. Individual subject numbers from which the viruses were isolated are shown in the legend. Subjects whose titers are represented with circles received the 2015–2016 flu vaccine, while those represented with triangles were not vaccinated. Experiments were repeated three times, showing reproducible data. IV, isolated virus. VV, vaccine virus. The LoD is indicated by the dotted line. Experiments were repeated 3 times, showing reproducible data.

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In patient sera, NAI Ab titers specific to the vaccine strain and the circulating strain significantly (P<0.001) increased at day 28 compared to day 0 (Fig 8A), strongly suggesting that natural infection induces production of NA-specific antibodies capable of inhibiting NA sialidase activity. NAI Ab titers in human sera specific to the vaccine strain were also significantly higher (with a 4-fold average difference) than NAI Ab titers specific to the circulating strain at day 0 and at day 28 in all 14 patients analyzed (P<0.0001). Furthermore, an 8-fold difference in strain-specific Ab titers was observed using ferret serum (Fig 8B). These results indicate that at least one of the mutations present in the NA protein of circulating strains contribute to the evasion of NA-Ab binding.