Fig. 4. IN spike boosting elicits enhanced mucosal immunity with similar systemic humoral responses to IM mRNA-LNP boosting.

(A) Experimental schema. K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed 14 days later by boosting with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike IN. Forty-five days after prime, lung tissues were collected for T cell analysis by means of flow cytometry, and BALF and serum were collected for antibody measurement. K18-hACE2 mice were intramuscularly primed with 0.05 μg of mRNA-LNP, followed 14 days later by boosting with 0.05 μg of mRNA-LNP intramuscularly, or 1 μg of SCV2 spike intranasally and challenged with 6 × 10⁴ PFU SCV2 at 118 days after prime. (B to D) Quantification of total tetramer⁺ CD8 T cells, CD69⁺CD103⁻tetramer⁺ CD8 T cells, or CD69⁺CD103⁺tetramer⁺ CD8 T cells in lung tissues from naïve, mRNA-LNP prime-boost, or P&S mice. (E to F) Quantification of total tetramer⁺ CD4 T cells or CD69⁺CD103⁻tetramer⁺ CD4 T cells in lung tissues. (G to K) Lung lymphocytes were isolated by means of Percoll gradient and restimulated with spike peptide megapool from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45⁻CD44⁺ CD4 T cells. (L to O) Measurement of SCV2 spike S1 subunit–specific (L) BALF IgA, (M) BALF IgG, (N) serum IgA, and (O) serum IgG in naïve, mRNA-LNP prime-boost, or P&S mice. (P) Measurement of neutralization titer against SCV2 spike–pseudotyped VSV. (Q to S) Weight loss, survival, and disease-free survival (<5% maximum weight loss) of mRNA-LNP prime-boost or P&S mice from 1 to 14 days after infection. (T and U) Measurement of infectious virus titer in lung and nasal turbinate tissues at 2 days after infection by means of plaque assay. To reduce overall number of experimental animals used, control data points from naïve and mRNA prime-boost are common to Figs. 4 and 6. Mean ± SEM. Statistical significance was calculated by means of [(B) to (O)] one-way ANOVA followed by Tukey’s correction or [(P), (T), and (U)] Student’s t test, and [(R) and (S)] log-rank Mantel-Cox test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and are pooled from two independent experiments.