**2.4 Application of the DNA-FACE™: development of a novel type of anti-SARS-CoV-2 vaccine**

The DNA-FACE™ concept of construction of "artificial", concatemeric protein with a vaccine functionality has been used to construct an anti-SARS-CoV-2 vaccine.

The co-polymerization type construct contains multiple copies of various epitopes clusters, derived from Spike and Nucleoprotein of the SARS-CoV-2 virus.

The clusters were selected using a proprietary method developed in BioVentures Institute Ltd. (Poland), which has also developed the DNA-FACE™ biotechnology.

The vaccine is the most "Frankenstein", as it is composed of: (*i*) N-terminal polyepitopic clusters of various amino acid sequences and (*ii*) protein adjuvant to enhance the immune response. The entire protein construct has a molecular weight of approximately 70 kDa only, as compared to Spike protein (140.3 kDa kDa) and Nucleoprotein (45.6 kDa). Nevertheless, immunogenicity and toxicity studies in animals (rabbits) have shown that the protein is not toxic and induces a strong, specific immune response (**Figure 9**). Additional advantages of such composite vaccine are: (*i*) no need for refrigeration, as the antigen does not contain native conformation protein, which has to be protected from denaturation, and (*ii*) potential for rapid module exchange if a new virus variant contains changes epitopes. Panel (a) shows a high level of biosynthesis of the recombinant anti-SARS-CoV-2 vaccine protein in *E. coli*. The recombinant gene was cloned into pETAMP1-A amplificationexpression vector. Upon induction with IPTG (**Figure 9A, lane 2**), a dominant band of approximately 70 kDa becomes evident, as compared to control, uninduced recombinant *E. coli* culture (**Figure 9A, lane 1**). The induced sample was subjected to western blotting, using primary anti-His6-tag antibodies (**Figure 9B**, **lane 1**) or rabbit anti-anti-SARS-CoV-2 vaccine (**Figure 9C,** lanes 1 and 2). In both lines, multiple bands appear, which is expected, as rabbits carry also their own anti-*E. coli* proteins antibodies. The band pattern in the lane 2 is different - the dominant protein band of approximately 70 kDa comprises the anti-SARS-CoV-2 vaccine, the remaining bands are most probably a mixture of the vaccine degradation products, reacting *E. coli* proteins, including those, which are co-induced with IPTG. Further assay included western blotting using native Spike and Nucleoprotein proteins (purchased from an independent, foreign company), expressed in human cells,

#### **Figure 9.**

*Biosynthesis and immunogenicity of the recombinant anti-SARS-CoV-2 vaccine. Lane M, Page RulerTM Plus Prestained Protein Ladder (Thermo Scientific) (a) SDS-PAGE analysis of the recombinant anti-SARS-CoV-2 vaccine protein biosynthesis in E. coli.; lane 1, control E. coli lysate, without induction of the recombinant gene expression; lane 2, E. coli lysate, 3 hours after induction of the recombinant gene expression. (b) Western blotting and immunodetection of the recombinant anti-SARS-CoV-2 vaccine protein using anti-His-tag antibodies. (c) Western blotting and immunodetection of the recombinant anti-SARS-CoV-2 vaccine protein, using antibodies purified from the immunized rabbit serum. Lane 1, E. coli lysate, 3 hours after induction; lane 2, purified anti-SARS-CoV-2 vaccine protein; lane Spike, immunodetection of human, recombinant SARS-CoV-2 Spike protein; lane Nucl., immunodetection of the human, recombinant SARS-CoV-2 Nucleocapsid protein.*

thus identical to those present in the virus, including posttranslational modification, absent in *E. coli.* Nevertheless, strong, specific immunological reaction was obtained. Currently, this novel type of anti-SARS-CoV-2 vaccine undergoes further full-scale evaluation, the regulated pre-clinical animal and *in vitro* tests and its clinical tests will follow shortly. Important aspect of this vaccine design is that it does not contain intact Spike and Nucleoprotein, which are known to be toxic to human immunological system, among other negative effects. Thus, it is expected, that the vaccine will have much reduced, if any, side effects upon vaccination. It needs to be emphasized that the DNA-FACE™ concept used to develop anti-SARS-CoV-2 vaccine also applies to essentially all types of microbial pathogens as well as to cancer cells.

### **3. Conclusions**

The DNA-FACETM biotechnology was developed for the construction of "artificial", repetitive genes, encoding concatemeric RNAs and proteins of any nt and aa sequence. The DNA-FACETM is capable of formatting of ordered polymers in a controlled process, containing 500 or more copies of DNA, RNA, or peptide repeats within a concatemer.

The constructed concatemeric genes yield efficient genetic expression of concatemeric proteins, which were tested in the development of:

i.New generation of vaccines with an enhanced stimulation of the immune system, including anti-SARS-CoV-2 vaccine.

*DNA-FACE™ - An* Escherichia coli*-based DNA Amplification-Expression Technology… DOI: http://dx.doi.org/10.5772/intechopen.101640*


The DNA-FACETM technology is uniquely suited for wide applications in the scientific research, biotechnology, pharmaceutical, and medical industries. The method goes far beyond current chemical genes synthesis capabilities. It allows for gene and protein design solutions, which were impossible before the development of this technology. This opens new research avenues not only for studying biological systems but also for practical solutions, such as novel types of cancer inhibitors, currently under development, using DNA-FACETM. We believe that the scientific and industrial community will recognize the potential of the DNA-FACETM technology, and several new applications of the technology will soon appear.
