Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies.

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry.

In this study, we constructed plasmids to increase the overall expression level of the SARS-CoV-2 S-protein and its presentation on the cell surface. To this end, we designed a series of plasmid constructs encoding the SARS-CoV-2 S-protein with modifications to its cytoplasmic domain and containing various 5′ and 3′ untranslated regions. Our results confirmed the critical role of the S-protein cytoplasmic domain in limiting its localization to the cell surface. We confirmed that deletion of the 19 C-terminal amino acids, which contain an endoplasmic reticulum retrieval signal, significantly increased S-protein presentation on the cell surface. Furthermore, introducing the HuR-binding site from the CD47 3′ untranslated region and replacing the 19 C-terminal amino acids of the S-protein with the CD47 cytoplasmic tail significantly enhanced total S-protein expression compared to the wild-type S-protein and constructs with the 19-amino-acid deletion. Unfortunately, for the plasmid constructs bearing CD47 elements, their higher surface expression compared to the wild-type S-protein correlated with a high total protein expression level.