A Study of the Influence of Nucleotide Sequence Optimization of the Target Gene on the Immunogenicity of a Recombinant Adenovirus Vector

This work highlights the importance of nucleotide sequence optimization of a target gene in the development of vaccines. The purpose of the study was to compare the immunogenicity of recombinant adenoviruses expressing the S glycoprotein gene of the SARS-CoV-2 Omicron (BA.5.3.1) variant with an optimized (Ad5-S-BA5opt) or nonoptimized (Ad5-S-BA5) nucleotide sequence. Recombinant adenoviruses were grown on HEK293 cell culture and purified by ultracentrifugation in a stepwise CsCl density gradient. The resulting adenovirus preparations were characterized by the authenticity (by real-time PCR and immunoblotting), the number of viral particles (spectrophotometrically), and the infectious titer (by endpoint dilution titration TCID₅₀). The titer of RBD-specific IgG antibodies in the blood serum of immunized animals was assessed by enzyme-linked immunosorbent assay (ELISA). Based on the results of studies in an animal model, a vector expressing the optimized gene (Ad5-S-BA5opt) resulted in the formation of higher levels of IgG-specific antibodies compared with a similar vector containing the wild-type protein gene. It is particularly interesting that an approach aimed solely at optimizing the nucleotide sequence of the target gene, and not other vector components, can significantly contribute to changes in the immunogenicity. The obtained results are important for the development of new, more effective strategies for creating vaccines and gene therapy drugs. Optimization of the nucleotide sequence can lead to an improved mRNA stability and a significant increase in translational efficiency and, as a consequence, to enhanced protective properties of the candidate drug. It can be supposed that this method will be promising for the development of vaccines against viruses containing low-immunogenic antigens.