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. 2023 Oct 25;8:161. doi: 10.1038/s41541-023-00755-2

Fig. 6. Immunogenicity of lyophilized RS2, that had been incubated at 37 °C for 1 month, in hACE-2 expressing transgenic mice.

Fig. 6

hACE-2 expressing transgenic mice immunized twice with 20 μg of lyophilized RS2 that was previously incubated at 37 °C for over a month and then formulated in SWE adjuvant. This was followed by intranasal challenge with 104 pfu Beta and Delta variants. a ELISA endpoint titers against RBD, S2, and spike ectodomain. b Neutralizing antibody titers against B.1, Beta, Delta and BA.1 pseudoviruses. c Neutralizing antibody titers elicited by lyophilized RS2 subjected to 37 °C for over a month (Lyo), and non-lyophilized RS2 (Non-lyo) stored at 4 °C, against B.1 and BA.1 Omicron SARS-CoV-2 pseudovirus (d, e) Average weight change up to nine days post-Beta and Delta virus challenge respectively. f Survival curve. g, h Lung viral titers in RS2 immunized mice, challenged with Beta VOC and Delta VOC respectively. i, j Histopathology scores of lungs. k Histology of lung tissue sections from unimmunized-unchallenged control (UC), mice immunized with 20 μg RS2 challenged with Beta variant (RS2-Beta challenged), unimmunized Delta virus challenged control (Unimmunized-Delta), mice immunized with 20 μg RS2 and challenged with Delta variant (RS2-Delta challenged), at 4X magnification. The scale bar indicates 50 µm. None of the unimmunized controls survived the Beta virus challenge (Unimmunized-Beta). Titers are shown as geometric mean with geometric SD. The ELISA binding, neutralization titer, lung viral titer and histopathology score data were analyzed with a two-tailed Mann–Whitney test. Neutralization titers for individual sera against B.1, Beta, Delta and BA.1 pseudoviruses (6b) were analyzed with non-parametric Kruskal–Wallis test with Dunn’s multiple correction. Weight changes were analyzed with a Multiple Student’s t test with Bonferroni Dunn’s correction method. (ns indicates non-significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).