Intranasal administration of RSV antigen-expressing MCMV elicits robust tissue-resident effector and effector memory CD8+ T cells in the lung

Abstract

Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T-cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T-cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T-cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T-cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability.

Figure 1. MCMV-M generates an M-specific CD8+ T cell population that inflates over time

Mice were infected with 2×10⁶ PFU of RSV via the intranasal (IN) route or 6×10⁵ PFU of MCMV-M via the IN or intraperitoneal (IP) route. At week 1 (W1) and week 6 (W6) post-infection, the percentage of M-specific CD8+ T cells in the lungs was determined using tetramer staining and flow cytometry. Bars represent mean ± SEM with 5 mice per group. **** p≤0.0001 and **p<0.01 by two-way ANOVA with Tukey’s post-test for multiple comparisons. Data represent two independent experiments.

Figure 2. IN vaccination generates more M-specific CD8+ T cells in the lung parenchyma than IP vaccination

At weeks 1 (W1), 6 (W6), 16 (W16), and 24 (W24) post-vaccination with MCMV-M via the IN or IP route, mice were injected IV with anti-CD45 antibody 5 minutes prior to sacrifice to identify cells in the blood (black) or tissue (grey). M-specific CD8+ T cells were identified by tetramer staining and flow cytometry. (a, b) Percentage of M-specific CD8+ T cells in the tissue and blood of the lungs (a) and spleen (b). (c) Expression of CD69 and CD103 on M-specific CD8+ T cells in the lung tissue at 16 weeks post-vaccination. The percentage of CD8+ T cells in each quadrant is indicated. (d) Total number of CD69+CD103+ and CD69+CD103- M-specific CD8+ T cells in the lung tissue at 16 weeks post-vaccination with MCMV-M or infection with RSV. e) Expression of CD69 and CD103 on M-specific CD8+T cells in the lung tissue at 24 weeks post-vaccination. f) Total number of CD69+CD103+ and CD69+CD103- M-specific CD8+ T cells in the lung tissue at 24 weeks post-vaccination with MCMV-M. Bars represent mean ± SEM with 5 mice per group. **** p≤0.0001 by two-way ANOVA with Tukey’s post-test for multiple comparisons. Data represent two independent experiments.

Figure 3. IN vaccination with MCMV-M augments the M-specific CD8+ T cell response via the induction of effector and effector memory cells in the lung parenchyma and blood

The memory phenotype of M-specific CD8+ T cells harvested from the indicated sites was determined at week 1 and week 6 post-vaccination with MCMV-M. Mice were injected IV with anti-CD45 antibody 5 minutes prior to sacrifice to identify cells in the blood or tissue. (a) Total number of central memory (CM), effector memory (EM), effector (E), and KLRG1⁺ effector (KLRG1+) cells at week 1 post-vaccination. (b) Total number of CM, EM, E, and KLRG1+ cells at week 6 post-vaccination. Bars represent mean ± SEM with 5 mice per group. **** p≤0.0001, *** p<0.001, **p<0.01, and *p<0.05 by two-way ANOVA with Tukey’s post-test for multiple comparisons. Data represent two independent experiments.

Figure 4. Large volume IN vaccination leads to increased viral replication in the lung and is necessary for the generation of tissue-tropic M-specific CD8+ T cells

(a) Mice were vaccinated IN with 1.2×10⁵ PFU of MCMV-M in a total volume of 20μl, 50μl, or 100μl. The percentage of M-specific CD8+ T cells in the lung at 6 weeks post-vaccination was determined using tetramer staining and flow cytometry. Bars represent mean ± SEM with 5 mice per group. (b) Quantification of MCMV genome copy number by qPCR in the lung, spleen, and salivary glands of mice infected IN or IP with 6×10⁵ PFU of MCMV-M in 100μl. Error bars represent SEM. Dotted line indicates limit of detection. ****p<0.0001 and ***p<0.001 by two-way ANOVA with Tukey’s post-test for multiple comparisons.

Figure 5. IN vaccination with MCMV-M leads to earlier anti-viral responses compared to IP vaccination

Mice were challenged with 2×10⁶ PFU of RSV either in the absence of prior vaccination or 16 weeks after IN or IP administration of MCMV or MCMV-M. On days 2-5 post infection, the lungs were harvested for plaque assay and cytokine analysis. (a) Viral loads on day 5 post-infection determined by plaque assay. Dashed line indicates limit of detection. (b) Viral loads on day 5 post-infection after CD8+ T cell depletion. Dashed line indicates limit of detection. NS = not significant. (c) Serial viral loads after challenge with RSV. Dashed line indicates limit of detection. Error bars indicate SEM. Asterisks indicate significant differences between MCMV-M IN and MCMV-M IP. (d) Viral loads on day 5 post-infection after FTY720 treatment. Dashed line indicates limit of detection. (e, f) Concentration of IFNγ (e) and MIP-1β (f) in the lungs determined by multiplex bead-based array after challenge with RSV. Asterisks indicate significant difference between MCMV-M IN and MCMV-M IP. ****p<0.0001, ***p<0.001 and *p<0.05 by one-way ANOVA with Tukey’s post-test for multiple comparisons.