Fig. 3: Pseudovirus neutralizing titers of serum samples collected from vaccinated mice.

Transgenic hACE2-expressing mice, (n = 6 per group, 3 males and 3 females), were immunized with different mRNA formulations administered intradermally at two-week intervals. The mRNA group received 20 μg of consensus spike mRNA (mRNA), the Trans-amplifying mRNA: high dose (TAHD) received 0.5 μg of consensus spike mRNA combined with 20 μg of replicase mRNA, and the Trans-amplifying mRNA: low dose (TALD) group received 0.05 μg of consensus spike mRNA with 20 μg of replicase mRNA. Mock-vaccinated mice received an equivalent volume (50 μL of phosphate-buffered saline, PBS). Serum samples were collected on day 28 and subjected to a neutralization assay using various SARS-CoV-2 pseudoviruses. The effective dilution 50% (ED₅₀) values were derived from the dose-response curves using a four-parameter Hill equation to assess pseudovirus neutralization efficacy. A Serum from mice immunized with 20 μg of consensus spike mRNA exhibited antibody titers against all variants, with ED₅₀ values (serum dilution factor) ranging from 223.3 ± 13.5 (Omicron BA.1) to 468.8 ± 39.9 (B.1 ancestral variant). B Mice immunized with a ‘40-fold lower dose of consensus spike mRNA’ (0.5 μg) in combination with VEEV replicase (TAHD) also demonstrated antibody titers against all SARS-CoV-2 pseudovirus variants, with ED₅₀ values ranging from 131.3 ± 13.1 (Omicron BA.1) to 188.0 ± 27.0 (B.1 ancestral variant). C Immunization with a low dose of consensus spike mRNA (0.05 μg) combined with VEEV replicase (TALD) resulted in minimal neutralization efficacy. ED₅₀ values were not calculated for this group, as the highest neutralization responses against all pseudovirus variants were not substantial enough to compute ED₅₀ values with a high degree of confidence.