Rabies is deadliest viral infection with nearly 100% fatality. There is currently no effective treatment. The problem of rabies in Kazakhstan remains unresolved, natural foci of the disease are constantly recorded, which requires an increase in the effectiveness of rabies prevention and control measures. The only treatment for rabies is the use of specific prophylaxis with vaccines and anti-rabies immunoglobulin (RIG). The RIG is derived from the plasma either of horses or humans. Both products have several limitations relate to safety, supply, and cost.

Project goal:

Development of novel nanobodies for the efficient neutralization of RABV. Engineering highly specific and sensitive nanobody-derived probe for rapid detection RABV in the immunoassays.

Objectives of the project:

1. expression of RABV-G protein as correctly folded recombinant protein and evaluation of antigenicity of the expression product.

2. Construction of a cDNA-VHH library by phage display methods and selection of RABV-G protein specific nanobodies.

3. Study of RABV neutralization efficacy of mono-, bi- and trivalent RABV specific nanobodies on mice challenged by a lethal dose of RABV.

Results:

Human rabies poses a serious threat to the health of the population of Kazakhstan, natural foci of the disease are constantly recorded, which requires an increase in the effectiveness of rabies prevention and control measures. This project is aimed at creating new nanobodies to effectively neutralize the rabies virus, which has great potential.

In this project, specific nanoantibodies against the rabies virus were isolated from the blood serum of camels using reverse transcription and PCR methods. Various forms of nanoantibodies have been created that contribute to the effective neutralization of the rabies virus. The recombinant glycoprotein (G-protein) of the rabies virus was cloned and expressed in bacterial and yeast systems. A specific nanobody with alkaline phosphatase has been created, which makes it possible to accelerate the process of detection of the rabies virus in samples by enzyme immunoassay using a single step.

Thus, the development of specific nanobodies against the rabies virus can lead to a significant reduction in the cost of existing vaccines, solve the problem of supplying remote areas, and has great prospects for further commercial production.

Project goal:

The main goal of the project is to decipher and characterize at the molecular level the mechanisms involved in the aberrant BER pathway initiated by the mismatch-specific thymine-DNA glycosylase in vitro and in vivo, and to evaluate the physiological role of aberrant BER in the accumulation of spontaneous and damage-induced DNA mutations in living cells.

Project objectives:
  1. Detailed biochemical characterization of the monofunctional human DNA glycosylase TDG initiating the aberrant BER pathway to DNA duplexes containing complex DNA lesions in vitro.
2. In vitro recovery of aberrant DNA repair assay with purified DNA glycosylase.
3. Characterization of the cellular response to genotoxic stress in S. cerevisiae and E. coli strains overexpressing the human monofunctional DNA glycosylase TDG.

Results:

 
  DNA substrates containing oxidized bases, aristolactam-adenine adducts, exocyclic DNA adducts, and DNA-DNA crosslinks have been synthesized and purified. A recombinant plasmid with the cDNA of the hTDG gene under the control of the inducible T7 bacteriophage promoter and with the 6xHis•tag sequence at the N-terminus was constructed using genetic engineering methods, which allows affinity purification of the protein product based on metal-chelate chromatography. The functional expression of hTDG was carried out in the expression strain E. coli Rosetta (DE3) and the purification of the recombinant hTDG protein with the 6X His-terminus to a homogeneous state by metal-chelate chromatography was carried out. Catalytically inactive TDGN140A and hyperactive TDGA145G and TDG H151Q forms of hTDG were created by site-directed mutagenase. The substitution of AAC for HCA in the nucleotide sequence of the mutated hTDGN140A was confirmed by sequencing. Functional expression of 6xHis-labeled hTDG N140A in the Rosetta(DE3) expression strain and homogeneous purification of the recombinant inactive hTDG N140A protein by affinity chromatography were carried out. The substrate specificity of human TDG was characterized for oligonucleotide substrates with oxidized bases, exocyclic DNA adducts, and aristolactam-adenine adducts. Work is underway to compare the activity of full-length human TDG and TDG cat and its active site mutants against oligonucleotide substrates with oxidized bases, exocyclic DNA adducts, aristolactam-adenine adducts, and inter- and intrastrand DNA crosslinks.