2014 December 10 -- Agenda and attachments

<p>(A) The virus titer in the lung and (B) proportion and (C) number of each epitope-specific CTL population, and (D) the combined total number for the three epitope-specific CTL populations in the BAL. (E) Representative flow cytometry plots for each tetramer-specific CTL population in the BAL. Details of the data analysis and comparisons are same as shown in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004642#ppat.1004642.g006" target="_blank">Fig. 6</a>.</p

Diverse Heterologous Primary Infections Radically Alter Immunodominance Hierarchies and Clinical Outcomes Following H7N9 Influenza Challenge in Mice

<div><p>The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8+ cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K^bPB1₇₀₃, D^bPA₂₂₄, and D^bNP₃₆₆ epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.</p></div

Sequence identity of CTL epitope-associated peptides in the studied viruses.

<p>^a ∙ represents an amino acid residue identical to that in the first row.</p><p>Sequence identity of CTL epitope-associated peptides in the studied viruses.</p

Global expression profiling of lung tissue isolated from influenza-infected mice.

<p>A) Heatmap depicting 385 genes whose expression is altered in the lung during the first 24 hours following infection with influenza virus (permutation q-value=<0.001 and > 5-fold difference). B) Mean expression of all genes within each cluster (K means with k=5 for 385 genes) grouped by virus strain (X31 – green; PR8 – red; VN – blue). The number of genes in each cluster is indicated. Error bars depict the SEM for all genes in the cluster at each time point.</p

The disease course in naive or primed (with H1N1 or H9N2 IAVs 10–12 weeks previously) mice following challenge with the H7N9 virus.

<p>The mice were challenged with 10^4.5 TCID₅₀ H7N9 virus (10 MLD₅₀). (A) The survival ratio and (B) weight loss during the disease course. Data represent mean ± SEM, n = 8–10/group from two independent experiments. (A) * p<0.05, log-rank test, naive <i>versus</i> other four primed groups. (B) * p<0.05, t test, naïve <i>versus</i> the every other four primed groups; # p<0.05, t test, Ck/HK/TP38 <i>versus</i> the every other three primed groups.</p

Tnf and Ly6i expression in PR8 or X31 infected LET-1 cells.

<p>LET-1 cells were infected with either PR8 (red) or X31 (green) and relative levels of endogenous (<b>A</b>) Tnf and (<b>B</b>) Ly6i transcripts determined from RNA isolated at specified time points. Error bars represent SEM for n=2.</p

The disease course and primary CTL responses in naive mice after infection with one of two H9N2 or two H1N1 IAVs.

<p>The primary IAV infection was conducted as described in the legends to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004642#ppat.1004642.g001" target="_blank">Fig. 1</a>. (A) Body weight change and (B) virus replication kinetics in mice. (C) The proportion and (D) number of each epitope-specific CTL population, and (E) the total number of the three epitope-specific CTLs in the BAL on d8 or d10. (F) Representative IFN-γ ICS flow cytometry plots from CD8+ T cells in BAL samples. The PB1₇₀₃, PA₂₂₄, and NP₃₆₆ peptide variants specific for each virus were used to stimulate the CTLs to produce IFN-γ for the ICS assay. The data sets represent mean ± SEM; * p<0.05 by Tukey’s test, comparing: (A, B) each virus <i>versus</i> every other virus at that time point, n = 10 per group; (C, D) the indicated epitope <i>versus</i> the other two epitopes in the virus, n = 4–5; (E) the indicated virus <i>versus</i> the other viruses.</p

Experimental design for analyzing CTL-mediated heterosubtypic immunity against H7N9 virus infection.

<p>8–10 week old female B6 mice were first primed intranasally with 10⁴ TCID₅₀ of an H9N2 virus or 10² TCID₅₀ of an H1N1 virus. The virus-specific primary CTL responses in the bronchoalveolar lavage (BAL) were characterized on day (d) 8 and/or d10 post inoculation (p.i). Blood was collected for Hemagglutination inhibition (HI) assays on d35. The virus-specific memory CTLs in the spleen were characterized on d38. Between 10~12 weeks after the initial priming, the primed mice were intranasally challenged with an H7N9 virus and the H7N9 virus-specific-secondary CTL response in the BAL was characterized on various days between d0 to d14 after challenge infection.</p

The disease course in young or aged naïve mice, or primed (with H9N2 or H1N1 virus) aged mice following H7N9 challenge.

<p>The aged females were between 16–18 months at priming and then were challenged about two months later or young (8–10 weeks). Aged matched or young (8–10 weeks) naïve female mice were used for comparisons. (A) The survival ratio and (B) body weight change of the mice after challenge with 10MLD50 of AH/1(H7N9) (data represent mean ± SEM, n = 5–6/group). (C) The body weight change, (D) virus titer in the lung, and (E) the total number of the three-H7N9 virus epitope-specific CTL populations in the BAL samples on d8 after challenge with 1MLD50 of AH/1(H7N9) virus (data represent mean± SEM, n = 3/group). (C, D, E) * p<0.05, t test, indicated group <i>versus</i> the naïve aged group.</p

Summary of virus-specific CTL epitope hierarchies.

<p>Summary of virus-specific CTL epitope hierarchies.</p