Fig. 1: Design aspects and protein expression of mRNA constructs in the trans-amplifying mRNA (TA mRNA) vaccine coding for SARS-CoV-2 consensus spike.

A The table shows pairwise comparisons between SARS-CoV-2 spike protein sequences, comprising a consensus sequence and nine variants. The upper-right triangle (blue) displays percentage identity values, while the lower-left triangle (white) demonstrates the number of amino acid differences between sequences. The consensus spike used in this study exhibited close similarity (98.82% to 98.35%) to the earlier SARS-CoV-2 variants (ancestral to Delta) and lower similarity (96.63% to 97.17%) to the later variants (Omicron BA.1/BA.2 to Omicron BA.5, as tested in the study). B Structural superimposition of the SARS-CoV-2 consensus spike protein (white) with the SARS-CoV-2 spike protein 6XKL (teal) was performed. The sequence alignment score was 5565.7, and the Root Mean Square Deviation (RMSD) between 745 pruned atom pairs was 1.154 Å, indicating high structural similarity. C The TA mRNA vaccine comprises two mRNA constructs: one encoding the replicase derived from the Venezuelan Equine Encephalitis Virus (VEEV), preceded by the 5’UTR from the HBA1 gene and followed by the 3’UTR from AES and mtRNR1; and another encoding the SARS-CoV-2 consensus spike protein, with sequences flanking it coding for conserved sequence elements (CSEs) of the VEEV replicate. D A schematic representation of the SARS-CoV-2 consensus spike protein highlights the sequence modifications relative to the ancestral B.1 lineage spike protein (GenBank: QHD43416.1). Modifications shown in grey represent consensus changes derived from variant alignments. Additional modifications were introduced to stabilize the spike protein in its pre-fusion conformation: three alterations at the S1/S2 cleavage site (shown in green) and six proline substitutions (shown in red). E A549 cells were transfected with mRNAs encoding the TA mRNA vaccine. Four hours post-transfection, total protein was subjected to western blot analysis using specific antibodies to detect protein expression. The consensus spike protein was observed in both full-length (180 kDa) and cleaved (78 kDa) forms, while the replicase appeared as a 53 kDa protein. Beta-actin (42 kDa) served as a reference control for the cells (Lane 1: Marker, Lane 2: Cell lysate).