Live attenuated vaccination protects aged chimeric ACE2 mice from severe SARS-CoV-2 pathogenicity in vivo
Fig 5
LAVNsp16 immunization induces local and systemic adaptive immune responses.
Eight weeks old chACE2 mice were infected with 7x104 PFU of wt virus or LAVNsp16. Bronchoalveolar lavage (BAL), serum, and lymphocytes derived from lung and spleen tissue were collected on day 21 postinfection. Spike-specific antibody levels in serum (A) and BAL fluid (B) were determined by ELISA. Mean luminescence + /- s.d upon HRP-coupled secondary antibody incubation is shown overlayed by data points of individual animals. S-specific B cells (C) and T cells (E, F) present in spleen or lung tissue were identified by antibody staining and subsequent flow cytometric analysis. Cell counts are displayed as mean + /- s.d. overlayed by individual data points. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test (B, D, E) or Kruskal-Wallis analysis followed by Dunn’s test (A, C). (D) Neutralization capacity of S-specific antibodies from sera of wt- or LAVNsp16-infected animals was analyzed in pseudotyped virus neutralization assays. Neutralization capacity is shown as mean inhibition of quadruplicate infections + /- s.d. (G, H) Spike-specific CD8+ T cells were further discriminated into KLRG1+ CD127- effector T cells (TEFF), KLRG1+ CD127+ effector memory T cells (TEM), KLRG1- CD127+ central memory T cells (TCM), and single or double positive CD69+ CD103+ tissue resident T cells (TRM). (I, K) Lung and spleen-derived T cells were restimulated ex vivo with S-derived peptides (VNFNFNGL and VTWFHAIHVSGTNGT). Resulting production of CD107a, IFNγ, TNFα, and IL2 by CD8+ T cells was determined by intracellular cytokine staining and analyzed by flow cytometry. Results are plotted as mean counts/million + /- s.d. Statistical analysis was done by two-way ANOVA followed by Bonferroni correction.