Transcriptomics in lung tissue upon respiratory syncytial virus infection reveals aging as important modulator of immune activation and matrix maintenance.

Aging poses an increased risk of severe infection by respiratory syncytial virus (RSV). The many different biological pathways comprising the response to infection in lungs that are influenced by aging are complex and remain to be defined more thoroughly. Towards finding new directions in research on aging, we aimed to define biological pathways in the acute response to RSV that are affected in the lungs by aging. We therefore profiled the full transcriptome of lung tissue of mice prior to and during RSV infection both at young and old age. In the absence of RSV, we found aging to downregulate genes that are involved in constitution of the extracellular matrix. Moreover, uninfected old mice showed elevated expression of pathways that resemble injury, metabolic aberrations, and disorders mediated by functions of the immune system that were induced at young age only by an exogenous trigger like RSV. Furthermore, infection by RSV mounted stronger activation of anti-viral type-I interferon pathways at old age. Despite such exaggerated anti-viral responses, old mice showed reduced control of virus. Altogether, our findings emphasize important roles in aging-related susceptibility to respiratory disease for extracellular matrix dysfunctions and dysregulated immune activation in lungs

Identification of Novel Avian Influenza Virus Derived CD8+ T-Cell Epitopes

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development

Novel AIV-specific CD8+ T cell epitopes within the LPAI H7N1 virus.

<p>Epitope mapping resulted in 11 novel CD8+ T-cell epitopes in the nucleoprotein (A) and 5 epitopes in the matrix 1 protein (B) of the LPAI H7N1 virus. In black, B12-restricted epitopes, in grey, B4-restricted epitopes (A) or B19-restricted epitopes (B).</p

CD8+ T-cell frequencies in tissues of LPAIV infected chickens.

<p>Percentages of CD8αα+CD3+ T cells (A–C) and CD8βα+CD3+ T cells (D–F) were analysed by flowcytometry in lung, spleen and PBMC at several days post infection. Absolute numbers were calculated by multiplying the percentage of CD8αα+CD3+ T or CD8βα+CD3+ T cells with the total number of cells isolated from lung (G, I) and spleen (H, J). Mean plus SEM is shown. In white: uninfected controls (UNINF, n = 12), in grey infected birds (n = 3 per time point).</p

Identification of MHC B12-restricted CD8+ T-cell epitopes using peptide pools.

<p>Lung cells were stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis. Results for three individuals birds are shown at 5 dpi (A–C), 7 dpi (D–F), 10 dpi (G–I) and 14 dpi (J–L). Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

Epitope prediction for MHC B12 based on anchor residues.

<p>Predicted epitopes and their localization based on anchor residues that have been described for B12 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Kaufman1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Wallny1" target="_blank">[33]</a>. X represents any amino acid. Anchor residues specific for the different MHC types are indicated in bold. A variable number of amino acids between the anchor residues is indicted with (X).</p

Screening of MHC B12-restricted CD8+ T-cell epitopes using individual peptides.

<p>Lung cells were isolated, <i>in vitro</i> re-stimulated with B12-restricted peptide pools and IFNγ-producing cells were determined by IFNγ ELIspot analysis at 7 dpi (A) and 10 dpi (B). Mean plus SEM is shown, n = 4 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

Identification of MHC B4-, B15-, B19- and B21-restricted CD8+ T-cell epitopes using peptide pools.

<p>Lung cells isolated at 10 dpi were stimulated with B4 restricted peptide pools (A) or, B15 (B), B21 (C) and B19-restricted peptide pools (D–F) and IFNγ producing cells were determined by IFNγ Elispot analysis. Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p

Epitope prediction for MHC B4, B15, B19 and B21 based on anchor residues.

<p>Predicted epitopes and their localization based on anchor residues that have been described for B4, B15, B19 and B21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Kaufman1" target="_blank">[32]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031953#pone.0031953-Koch1" target="_blank">[34]</a>. X represents any amino acid. Anchor residues specific for the different MHC types are indicated in bold. A variable number of amino acids between the anchor residues is indicted with (X).</p

Identification of MHC B4-restricted CD8+ T-cell epitopes using individual peptides.

<p>Lung cells isolated at 10 dpi were thawed and re stimulated with B4 restricted peptides and IFNγ producing cells were determined by IFNγ Elispot analysis. Mean plus SEM is shown, n = 3 per group. Positive responses (*) and “significant” peptides inducing a positive response in 2 out of 3 chickens (↓) are indicated.</p