Establishing H1-specific CD4 T cell memory prior to secondary challenge partially restores the H1-specific antibody response.

<p><b>A:</b> Mice were infected and immunized as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176407#pone.0176407.g003" target="_blank">Fig 3</a> and serum antibody responses were measured by H1 HA ELISA assay at day 8 following secondary infection. Data demonstrate the average OD from serum obtained from between 8–10 individual mice in the X31-HA, X31-HA-x139, and X31-sham-x139 groups and 3 individual mice in the naïve and sham-x139 groups, with error bars depicting the standard error of the mean. ** = p<0.01 using a linear mixed effect model for pairwise group comparisons. <b>B:</b> Mice were infected and immunized as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176407#pone.0176407.g003" target="_blank">Fig 3</a> and serum neutralizing antibody responses were determined by microneutralization assay at day 24 following secondary infection. Squares represent the neutralizing antibody titer in individual mice, with the geometric mean of the microneutralization titer shown by a line and error bars depicting the 95% confidence interval. * = p<0.05 by Wilcoxon rank-sum test.</p

The suppression of novel responses demonstrated following a secondary influenza infection is associated with a marked decline in antigenic load.

<p><b>A:</b> Mice were infected with 300,000 EID₅₀ of X-31 (H3N2) influenza and then were rested for 8 weeks. These mice, together with a cohort of naïve mice, were then infected with 50,000 EID₅₀ of x139 (H1N1) influenza. Mice only infected with X-31 8 weeks prior served as a control for waning immunity. CD4 T cell responses in splenocytes derived from 3 to 4 individual mice per group were quantified at day 8 post x139 infection by IL-2 Elispot assay, with data presented as the average spot count per million CD4 T cells after subtracting background. Error bars represent the standard error of the mean. <b>B:</b> Sera from these same 3 to 4 individual mice per group were pooled and the titer of HA-specific antibodies in each group was quantified using an HA ELISA assay, with data presented as the average of duplicate wells. <b>C:</b> Viral load in the lungs of 3–4 individual mice per group was determined by quantitative PCR using primers and probe derived from the M1 protein at days 2, 4, and 8 following x139 infection. Samples were run in quadruplicate, with the average qC for each sample determined and plotted on a standard curve. Data are presented as the average number of copies per mL, with error bars representing the standard error of the mean.</p

H3-specific antibody responses are not boosted following secondary infection with the x139 H1N1 virus.

<p>To evaluate for evidence of original antigenic sin, serum from mice in each group was tested for antibody against the A/Brisbane/10/07 H3 HA protein by ELISA assay at 24 days following x139 viral challenge. Data represent the average OD obtained at a given serum dilution from 5–6 individual mice per experimental group, with the exception of the sham-x139 group where only 2 mice were examined. There were no statistically significant differences between H3 antibody levels in the sham-immunized (X31-sham-x139) or HA-immunized (X31-HA-x139) mice when compared to antibody in the mice initially primed with the X31 virus and immunized with HA peptides without a subsequent H1 viral infection (X31-HA).</p

I-A^s peptide-epitope alignment¹.

<p>I-A^s peptide-epitope alignment<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176407#t001fn001" target="_blank">¹</a>.</p

Immunization with a pool of HA peptides restores CD4 T cell reactivity to the selected HA epitopes following a secondary infection.

<p>Mice were infected and immunized as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176407#pone.0176407.g003" target="_blank">Fig 3</a>. At day 8 following the secondary infection, individual spleens and mediastinal lymph nodes were harvested. CD4 T cells were isolated by MACS cell purification and reactivity to a panel of peptide-epitopes was measured by IL-2 Elispot assay. <b>A:</b> CD4 T cell reactivity in the spleen. Data represent results from 9–13 individual mice averaged in each experimental group. <b>B:</b> CD4 T cell reactivity within the mediastinal lymph node. Data presented represent the average results from 6–13 individual mice in each experimental group. All data are presented as the spot count normalized to 10⁶ CD4 T cells after subtracting background, with error bars depicting the standard error of the mean. * = p<0.05; ** = p<0.01; *** = p<0.001 by Kruskal-Wallis one-way ANOVA test.</p

Reactivity following HA peptide priming is not influenced by previous X-31 infection.

<p>A cohort of B10.S mice was infected with 300,000 EID₅₀ of X-31 (H3N2) influenza and rested for 4 weeks. These previously infected mice, together with a cohort of naïve mice, were then immunized intraperitoneally with a pool of 5 H1 HA peptides in alum as described. Spleens were harvested at day 10 post immunization and CD4 T cells were isolated by MACS cell purification, with reactivity to a panel of selected epitopes determined by Elispot assay. <b>A:</b> Reactivity to selected peptide-epitopes as measured by IL-2 Elispot assay; <b>B:</b> Reactivity to peptide-epitopes quantified by IFNγ Elispot. Data represent the results obtained from 3–6 individual mice per experiment group, with the spot count normalized to 10⁶ CD4 T cells after subtracting background and then averaged.</p

Experimental design to examine the effect of establishing HA-specific memory by peptide immunization prior to secondary infection.

<p>B10.S mice were infected with 300,000 EID₅₀ of X-31 (H3N2) influenza virus and were rested for four weeks. Groups 1 and 3 were then immunized with a pool of 5 HA peptides in alum IP as described while Group 2 and 4 were only immunized with alum in PBS (sham). After 4 weeks, Groups 1, 2 and 4 were infected with 50,000 EID₅₀ of x139 (H1N1) influenza virus. Spleen, mediastinal lymph nodes, and serum were harvested at days 8 or 24 following secondary infection (Group 4 only had serum harvested).</p

The B cell response following infection with different viruses that replicate in the lung.

<p>(A–F) Virus-specific ASC frequencies. DR1 (A–C) and B10 (D–F) mice were infected intranasally with the influenza viruses PR8 (A and D) and X31 (B and E), and with the non-influenza virus MHV68 (C and F). Virus-specific ASC frequencies in the MedLN were determined by ELISPOT assay at intervals after infection. (G) PR8 replication in the lung. Titers are represented as log₁₀ TCID₅₀/0.2 ml of lung homogenate. (H, I) Flow cytometric analysis of ASCs and germinal center B cells in the MedLN on day 8 after PR8 infection. ASC frequencies (H) represent the proportion of B220^int CD138⁺ cells after gating on live CD4⁻ CD8⁻ CD19⁺ cells. Germinal center B cell frequencies (I) represent the proportion of PNA⁺ Fas⁺ cells among live CD4⁻ CD8⁻ B220⁺ cells. (J) Serum levels of virus-specific IgG in DR1 and B10 mice on day 8 after PR8 infection. Titers determined by ELISA are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g006" target="_blank">Fig. 6</a> data sets depict the mean+SE for 3–10 individual mice per group. * P<0.05, *** P<0.001.</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

Cytokine and chemokine production in the lung after influenza infection.

<p>DR1 and B10 mice were infected intranasally with NC or PR8 or were mock-infected with PBS. Mice were sampled 60 h after inoculation. Cytokine and chemokine concentrations in clarified lung homogenates were determined by Multiplex assay or by sandwich ELISA (IFN-α only). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g009" target="_blank">Figure 9</a> shows the results for a selection of the 30 cytokine and chemokine determinations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> for the remaining cytokines and chemokines are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone.0034377.s004" target="_blank">figure S4</a>. The mean+SE is shown for 5 individual mice per group. * P<0.05, ** P<0.01, *** P<0.001.</p