Immunogenicity of recombinant fragment of orthopoxvirus p35 protein in mice

Despite the elimination of smallpox, orthopoxviruses continue to be a source of biological danger for humans, as cowpox and monkey pox viruses circulate in nature and the last virus can cause both sporadic cases of human diseases and outbreaks of smallpox-like infection. In addition, periodic vaccination is necessary for representatives of some professions (scientists studying pathogenic orthopoxviruses, medical personnel, etc.). Vaccination against smallpox virus with live vaccinia virus, which was widely used during the elimination of smallpox, induces the formation of long-term immunity in vaccinated people. However, providing a high level of protection, the vaccination is often accompanied by serious post-vaccination complications, the probability of which is particularly great for individuals with compromised immunity. In this regard, the development of preparations for the prevention and treatment of infections caused by orthopoxviruses remains important today. The aim of this study was to assess the immunogenicity in the mouse model of recombinant protein р35Δ12, designed previously on the base of the cowpox virus protein p35. It was previously shown that the protein р35Δ12 was recognized by fully human neutralizing anti-orthopoxviral antibody with high affinity. In this work, recombinant protein р35Δ12 produced in E. coli cells XL1-blue and purified by chromatography was used for two-time immunization of mice. Two weeks after the second immunization, blood samples were taken from mice and serum antibodies were analyzed. It was shown by ELISA and Western-blot analysis that immunized mice sera contained IgG antibodies specific to recombinant protein р35Δ12. Confocal microscopy showed that antibodies induced by the р35Δ12 protein were able to recognize Vero E6 cells infected with the LIVP-GFP vaccinia virus. In addition, the antibodies in the serum of immunized mice were able to neutralize the infectivity of the vaccinia virus LIVP-GFP in the plaque reduction neutralization test in vitro. These experiments have demonstrated promising properties of the р35Δ12 protein if it were used as a component of vaccine for prophylaxis of orthopoxvirus infections

Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus

Fully­human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full­size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully­human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO­S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR­CH­CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT­DHFR­CH­CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT­DHFRCH­CL cassette plasmid. Then, CHO­S/FRT cells, which contain the FRT­site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT­site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti­vaccinia virus activity in the plaque reduction neutralization test in vitro

ANALYSIS OF DOMAIN SPECIFICITY OF THE PROTECTIVE CHIMERIC ANTIBODY ch14D5a AGAINST GLYCOPROTEIN E OF TICK-BORNE ENCEPHALITIS VIRUS

A drug for the prevention and therapy of tick-borne encephalitis virus is being developed on the basis of the protective chimeric antibody ch14D5a. At the same time, the epitope recognized by this antibody on the surface of glycoprotein E has not been localized yet. The aim of this work was to identify the domain of glycoprotein E, to which the protective antibody ch14D5a binds. As a result, four recombinant variants of glycoprotein E were generated using the bacterial expression system: (1) the rE protein containing the domains D1, D2, and D3 of glycoprotein E; (2) the rED1+2 protein containing domains D1 and D2; (3) the rED3_301 protein, which is domain D3 of glycoprotein E, and (4) the rED3_294 protein comprising domain D3 and a hinge region connecting domains D1 and D3. The rED3_294 and rED3_301 proteins were obtained in soluble monomeric form. The rE and rED1+2 proteins were extracted from the inclusion bodies of Escherichia coli. Using Western blot analysis and surface plasmon resonance analysis, it was demonstrated that the protective chimeric antibody ch14D5a and its Fab fragment bound specifically to domain D3 of glycoprotein E. Since the antibodies recognizing epitopes on the surface of domain D3 do not tend to cause antibody-dependent enhancement of the infection as compared to antibodies directed to domains D1 and D2, the data obtained confirm the promise of using the antibody ch14D5a in the development of a therapeutic preparation against the tick-borne encephalitis virus