Fig. 4: Assessment of viral load in mice following SARS-CoV-2 BA.1 challenge post-vaccination.

Transgenic hACE2-expressing mice (n = 6 per group; 3 males and 3 females) were vaccinated twice, two weeks apart, intra-dermally with either PBS (mock), SARS-CoV-2 consensus mRNA (mRNA), or two different doses of a trans-amplifying mRNA vaccine: high dose (TAHD) or low dose (TALD). Two weeks post-vaccination, mice were challenged intra-nasally with 10⁴ TCID₅₀ of SARS-CoV-2 Omicron BA.1. Four days after the challenge, lung tissues were collected, homogenized, and analyzed for the presence of SARS-CoV-2 N1 viral gene by qPCR and for infectious virus titers by TCID₅₀ assay. A qPCR results showed a significant difference in N1 copy numbers among the groups (p = 0.0004, Kruskal-Wallis test followed by Wilcoxon Rank Test for pair-wise comparison). The mock group exhibited the highest N1 load, with significantly lower viral loads observed in the mRNA group (p = 0.0095) and TAHD group (p = 0.0022). No significant difference in viral load was found between the mock and TALD groups (p = 0.065). The mRNA group had the lowest N1 levels compared to both the TAHD group (p = 0.0095) and the TALD group (p = 0.0095). Additionally, the TAHD group had lower N1 levels compared to the TALD group (p = 0.022), indicating a dose-dependent response. However, the presence of the N1 gene does not necessarily indicate live virus presence, underscoring the importance of the TCID₅₀ assay as a true measure of infectious viral load. B Infectious virus titers from homogenized lung samples showed a significant difference among the groups (p = 0.00067, Kruskal-Wallis test followed by Wilcoxon Rank Test for pair-wise comparison). The mock group had the highest levels of infectious virus, significantly differing from all other groups: mRNA (p = 0.034), TAHD (p = 0.022), and TALD (p = 0.0087). The mRNA group had infectious virus levels comparable to the TAHD group (p = 0.44), demonstrating that the TAHD vaccine was able to reduce the challenge virus to levels similar to those achieved with the mRNA vaccine, despite having 40 times less antigen mRNA load. The mRNA group had lower infectious virus levels compared to the TALD group (p = 0.024), and the TAHD group also had lower levels than the TALD group (p = 0.0022). The box plots display individual values with median values (thick black line in the middle) and 95% confidence intervals.