TY - JOUR AB - The potential of immune-evasive mutation accumulation in 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. The huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants has renewed interest in DNA vaccine technology. Here, we report the rapid generation and immunological evaluation of novel DNA vaccine candidates against the Wuhan-Hu-1 and Omicron variants based on the RBD protein fused with the Potato virus X coat protein (PVXCP). The delivery of DNA vaccines using electroporation in a two-dose regimen induced high-antibody titers and profound cellular responses in mice. The antibody titers induced against the Omicron variant of the vaccine were sufficient for effective protection against both Omicron and Wuhan-Hu-1 virus infections. The PVXCP protein in the vaccine construct shifted the immune response to the favorable Th1-like type and provided the oligomerization of RBD-PVXCP protein. Naked DNA delivery by needle-free injection allowed us to achieve antibody titers comparable with mRNA-LNP delivery in rabbits. These data identify the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study. AU - Dormeshkin, Dmitri AU - Katsin, Mikalai AU - Stegantseva, Maria AU - Golenchenko, Sergey AU - Shapira, Michail AU - Dubovik, Simon AU - Lutskovich, Dzmitry AU - Kavaleuski, Anton AU - Meleshko, Alexander ID - 13232 IS - 6 JF - Vaccines TI - Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein VL - 11 ER - TY - JOUR AB - Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. AU - Dormeshkin, Dmitri AU - Shapira, Michail AU - Karputs, Alena AU - Kavaleuski, Anton AU - Kuzminski, Ivan AU - Stepanova, Elena AU - Gilep, Andrei ID - 11462 JF - Applied Microbiology and Biotechnology SN - 0175-7598 TI - Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development VL - 106 ER -