Fig. 6: Co-immunization with S and N DNA vaccines provides long-term protection against SARS-CoV-2 Omicron BA.2 infection in K18hACE2 mice.

a Experimental protocol: On days 0 and 21, K18-hACE2 mice were inoculated with S and/or N DNA vaccines or pVAX control (25 µg, IM), challenged 17 weeks later with SARS-CoV-2 Omicron BA.2 (10⁴ PFU, IN), and tissues were harvested at 3 days post-challenge. Representation of the experiment timeline was Created with BioRender.com. b SARS-CoV-2 genomic and subgenomic RNA were quantified by RT-qPCR. Data are from 2 independent experiments (n = 9–10 mice/group). c Histopathology of SARS-CoV-2-induced lung disease. Five histopathological parameters (necrosis of bronchiolar epithelial cells [BEC], cellular debris in bronchioles, inducible bronchus-associated lymphoid tissue [iBALT] hyperplasia, perivascular lymphocytic cuffing, and bronchointerstitial pneumonia) were scored from 0 (least severe) to 5 (most severe). Data are from 2 independent experiments (n = 9–10 mice/group). d Representative bronchointerstitial pneumonia images, H&E-stain. e Splenocytes were stimulated with the indicated SARS-CoV-2 S or N peptides and polyfunctional (IFN-γ⁺TNF-α⁺ or IFN-γ⁺TNF-αIL-2⁺) and cytotoxic (IFN-γ⁺CD107a⁺) CD4⁺ and CD8⁺ T cells within the population of CD3⁺ cells were quantified by ICS. Data are from 2 independent experiments (n = 8–10 mice/group). f In vivo T cell depletion protocol (Created with BioRender.com): On days 0 and 21, K18-hACE2 mice were inoculated with S + N DNA vaccines or pVAX control (25 µg, IM). On days 137, 138, and 139, mice were injected with anti-CD4 and anti-CD8 or isotype control monoclonal Abs (250 µg each, IP), and then challenged on day 140 with SARS-CoV-2 Omicron BA.2 (10⁴ PFU, IN). Tissues were harvested 3 days later. Representation of the experiment timeline was Created with Biorender.com. g SARS-CoV-2 genomic and subgenomic RNA were quantified by RT-qPCR. Data are from 3 independent experiments (n = 8–9 mice/group). b, e, g Group means were compared by the non-parametric Kruskal-Wallis test (b, e) or non-parametric Mann–Whitney test (g). *P < 0.05, **P < 0.01. ***P < 0.001. Circles, individual mice; dashed lines (b, e), detection limit.