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

<p>(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.</p

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

<p>Mutations present in the NA protein of 2015–2016 circulating pH1N1-like strains compared to the vaccine strain.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188267#pone.0188267.s003" target="_blank">S2 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188267#pone.0188267.t001" target="_blank">Table 1</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188267#pone.0188267.s002" target="_blank">S1 Table</a>). 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) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188267#pone.0188267.ref062" target="_blank">62</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188267#pone.0188267.ref063" target="_blank">63</a>]. 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).</p

The CD4 T cell response to NC infection.

<p>(A–D) Cytokine production by peptide-specific CD4 T cells. DR1 (A and B) and B10 (C and D) mice were infected intranasally with 40,000 EID₅₀ NC. Enriched CD4 T cells from the MedLN and spleen were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (HA, NA, NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean+SEM for 2–6 independent experiments for each peptide. Cells from at least three mice were pooled for each experiment. (E, F) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A–D and represent 4 (MedLN) or 6 (spleen) independent experiments evaluating 3–12 (MedLN) or 12–20 (spleen) individual peptides. The mean+SEM is shown.</p

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

<p>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.</p

Modeling the Dynamics and Migratory Pathways of Virus-Specific Antibody-Secreting Cell Populations in Primary Influenza Infection

<div><p>The B cell response to influenza infection of the respiratory tract contributes to viral clearance and establishes profound resistance to reinfection by related viruses. Numerous studies have measured virus-specific antibody-secreting cell (ASC) frequencies in different anatomical compartments after influenza infection and provided a general picture of the kinetics of ASC formation and dispersion. However, the dynamics of ASC populations are difficult to determine experimentally and have received little attention. Here, we applied mathematical modeling to investigate the dynamics of ASC growth, death, and migration over the 2-week period following primary influenza infection in mice. Experimental data for model fitting came from high frequency measurements of virus-specific IgM, IgG, and IgA ASCs in the mediastinal lymph node (MLN), spleen, and lung. Model construction was based on a set of assumptions about ASC gain and loss from the sampled sites, and also on the directionality of ASC trafficking pathways. Most notably, modeling results suggest that differences in ASC fate and trafficking patterns reflect the site of formation and the expressed antibody class. Essentially all early IgA ASCs in the MLN migrated to spleen or lung, whereas cell death was likely the major reason for IgM and IgG ASC loss from the MLN. In contrast, the spleen contributed most of the IgM and IgG ASCs that migrated to the lung, but essentially none of the IgA ASCs. This finding points to a critical role for regional lymph nodes such as the MLN in the rapid generation of IgA ASCs that seed the lung. Results for the MLN also suggest that ASC death is a significant early feature of the B cell response. Overall, our analysis is consistent with accepted concepts in many regards, but it also indicates novel features of the B cell response to influenza that warrant further investigation.</p></div

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

<p>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.</p

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

<p>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.</p