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Inactivated influenza vaccines have struggled to provide consistent protection in older individuals. Circumventing immune senescence, an aging of the immune response characterized by weak humoral responses to vaccines, and unchecked inflammation during infection require novel immunization strategies. Plant-based virus-like particles (VLPs) bearing recombinant hemagglutinin proteins have been shown to provide protection in older animals in preclinical challenge studies, despite eliciting relatively low or absent humoral responses. The nature of the cellular response induced by these vaccines and its evolution during infection have not yet been fully characterized, however. Using a murine model that recapitulates features of human immune senescence, we assessed T cell responses to vaccination with a VLP bearing the hemagglutinin of H1N1/California 07/2009 (H1-VLP) before and after challenge in young and aged BALB/c mice (2 and 18 mo old, respectively). We report that two i.m. doses of H1-VLP (3 μg) vaccine 21 d apart generated H1-specific Th1 and Th2 cells associated with the prevention of prolonged pulmonary inflammation and mortality in both adult and aged mice. While investigating the regulation of cellular immunity, we identified a unique IL-1R1+ tissue-adapted regulatory T cell population in the lungs of both H1-VLP–vaccinated adult and aged mice, suggesting a novel regulatory T cell population associated with vaccine-mediated protection. Collectively, this study provides preclinical evidence that the plant-based H1-VLP vaccine may act, in part, by preventing exacerbated immune responses against influenza A.

Additional file 2: Figure S1. Flow cytometry-gating strategy for splenocytes. Aged (18–22 months) BALB/c mice were immunized twice with H1-VLP, split-virion vaccine or naïve. Three weeks post-boost (6–24 mice/group), splenocytes were collected and stimulated ex vivo for 18 h with H1-VLP.

Additional file 3: Figure S2. Higher dose IM + IN vaccination: Effects on lung immune response 42 days post-vaccination, viral load 3 dpi and survival after A/California/07/2009 challenge. Aged (18–22 months) BALB/c mice were immunized twice with H1-VLP, split-virion vaccine or PBS. Lungs were collected six weeks after vaccination. Percent of lung A) CD8+ T cells and expressing IFNγ presented as background subtracted (stimulated - unstimulated). Fold-change of tissue-resident B) CD4+ T cells from the PBS group. For statistical analysis, two-way ANOVA was performed followed by Tukey’s multiple comparison test (** p 20% of their initial weight. A log-rank (Mantel-Cox) test was used to compare survival curves with the PBS control group. (** p

Additional file 1: Table S1. Frailty Index measurements for day 0 and day 25 post-infection. Table S2. Statistics comparing groups for weight loss, using Tukeyâ s multiple comparison test. Table S3. Percent of mouse splenocyte T cells (CD4+ or CD8+) above the PBS average compared to the vaccinated animals

Additional file 3: Figure S2. Higher dose IM + IN vaccination: Effects on lung immune response 42 days post-vaccination, viral load 3 dpi and survival after A/California/07/2009 challenge. Aged (18–22 months) BALB/c mice were immunized twice with H1-VLP, split-virion vaccine or PBS. Lungs were collected six weeks after vaccination. Percent of lung A) CD8+ T cells and expressing IFNγ presented as background subtracted (stimulated - unstimulated). Fold-change of tissue-resident B) CD4+ T cells from the PBS group. For statistical analysis, two-way ANOVA was performed followed by Tukey’s multiple comparison test (** p 20% of their initial weight. A log-rank (Mantel-Cox) test was used to compare survival curves with the PBS control group. (** p

Additional file 2: Figure S1. Flow cytometry-gating strategy for splenocytes. Aged (18–22 months) BALB/c mice were immunized twice with H1-VLP, split-virion vaccine or naïve. Three weeks post-boost (6–24 mice/group), splenocytes were collected and stimulated ex vivo for 18 h with H1-VLP.