Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes.

Andrey Mazeika,Valery Shnyrov,Eduard Kostetsky,Nina Sanina,Vladimir N Uversky,Manuel Roig,Natalia Chopenko,Ludmila Davydova,Vasily Golotin

doi:10.3390/biom8030082

Abstract

Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection.

Highlights

Tick-borne encephalitis (TBE) is a viral infection that affects the main parts of the brain and spinal cord of the central nervous system, as well as spinal nerve roots and peripheral nerves
The recombinant plasmid 40HSP70 was obtained by ligation of the HSP70 gene into the pET-40b(+) at the restriction sites of NcoI and SacI
For all constructs analyzed in this study, the commercial vector pET-40b(+) was used. This plasmid has important features, such as the presence of the gene encoding redox protein DsbC, which promotes the correct protein folding of the recombinant protein. It has an N-terminal signal sequence, which allows the direction of the recombinant protein to the cell periplasm

Summary

Introduction

Tick-borne encephalitis (TBE) is a viral infection that affects the main parts of the brain and spinal cord of the central nervous system, as well as spinal nerve roots and peripheral nerves. A TBE infection may result in long-lasting neurological complications, or even death [1]. There is no specific antiviral medication against TBE infection, and the only currently available treatment for it is restricted to symptomatic therapy. TBE can be prevented successfully by vaccination. Available commercial vaccines based on the inactivated whole virus show mild and transitory side-effects [2]. This type of vaccine is successful, the manufacturing of inactivated

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