RESUMEN
The potential of immune evasive mutations accumulation of the SARS-CoV-2 virus has led to its rapid spread causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. Huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants renews interest in DNA vaccine technology. Here we report a rapid generation and immunological evaluation of novel DNA vaccine candidates against Wuhan-Hu-1 and Omicron variants, based on the RBD protein fused with the Potato virus X coat protein (PVXCP). Delivery of DNA vaccines using electroporation in a two-doses regimen induced high antibody titers and profound cellular response in mice. Antibody titers induced against Omicron variant of the vaccine were sufficient for the effective protection against both the Omicron and Wuhan-Hu-1 virus infections. PVXCP protein in the vaccine construct shifted immune response to the favorable Th1-like type and provided oligomerization of RBD-PVXCP protein. A naked DNA delivery by the needle-free injection device allowed us to achieve antibody titers comparable with the mRNA-LNP delivery in rabbits. This data identifies the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study.
RESUMEN
The recombinant truncated endolysin LysK consisting of two catalytic domains, N-terminal CHAP and amidase-2 (LysKCA) was overexpressed in E. coli in the form of inclusion bodies (IBs). These IBs were dissolved in 6 M solution of urea followed by the refolding process. The refolding efficacy of the dilution and matrix-assisted renaturation method on SP Sepharose was compared at different purification stages of LysKCA. Solubilizate of IBs, DEAE Sepharose flowthrough, and SP Sepharose elution fractions were examined. The presence of negatively charged nucleic acids (NA) in the solution has shown a decrease in the recombinant LysKCA refolding yield (less than 11.5 ± 1.3% for both renaturation methods) due to their non-specific interaction with the positively charged endolysin. The renaturation efficiency of the enzyme purified from NA (SP elution fraction) was about 29.5 ± 6.7% and 28.2 ± 3.75% for dilution and matrix-assisted methods respectively. The later approach allows conducting one-step LysKCA refolding, purification and collection, and also noticeably cuts time and material expenses. The analysis of CD spectroscopy data of LysKCA, renatured on the resin matrix, revealed alpha helices and beta strands content similar to that of the modeled 3D structure. The theoretical 3D model with two predicted domains (CHAP and amidase-2) agrees well with the differential scanning calorimetry (DSC) results of the renatured LysKCA showing two well-resolved peaks corresponding to the two calorimetrically-revealed domains with the midpoint transition temperature (Tm) of 40.1 and 65.3°Ð¡. The enzyme so obtained exhibited in vitro anti-staphylococcal activity with 2.3 ± 0.45 × 103 U/mg and retained it for at least one year.