Leonid R. Lebedev

Expression of genes for orthopoxviral TNF-binding proteins in insect cells and investigation of the recombinant TNF-binding proteins

Genes for TNF-binding proteins (CrmBs) of the variola virus (VARV), monkeypox virus (MPXV) or cowpox virus (CPXV) were isolated by PCR from viral genomes and expressed in a baculovirus system in the Sf21 insect cell line. Properties of the purified recombinant proteins were studied by various physicochemical and immunological methods. Using solid-phase enzyme-linked immunosorbent assay, it was shown that viral proteins inhibited hTNF binding with polyclonal anti-hTNF antibodies, with the efficiency of inhibition decreasing in the series VARV-CrmB > CPXV-CrmB > MPXV-CrmB. Biological activity of the recombinant protein preparations was assessed by their ability to neutralize TNF cytotoxicity on the L929 murine fibroblast cells line. CrmBs were shown to neutralize cytotoxicity of human, mouse, and rabbit TNF in a species-specific manner. It was also shown that the efficiency of VARV-CrmB in inhibiting hTNF cytotoxicity exceeded that of polyclonal anti-hTNF antibodies. Orthopoxviral CrmB proteins can provide a basis for development of new anti-TNF drugs.

Construction of artificial virus-like particles exposing HIV epitopes, and the study of their immunogenic properties

One of the major problems in the development of successful recombinant vaccines against human immunodeficiency virus (HIV) is that of correct identification of a safe and effective vaccine delivery system with which to induce protective immunity using soluble protein antigens. An original method for constructing artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface is reported. The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates. Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested.

Chimeric antibodies against tick-borne encephalitis virus

Two chimeric antibodies (ch) 13D6 and 10C2 against the glycoprotein E of tick-borne encephalitis virus (TBEV) were constructed by fusing variable regions of murine monoclonal antibodies (Mabs) 13D6 and 10C2 to human constant regions. Monovalent analogues of these antibodies in format of single-chain antibodies (scFv or sc) were developed, as well. The ch13D6, ch10C2, sc13D6 and sc10C2 exhibited binding characteristics similar to parental Mabs. Only the ch13D6 and sc13D6 were able to neutralize TBEV infectivity in vitro. The in vitro neutralization provided by ch13D6 suggests that this antibody can be further developed into a potent prophylaxis and therapy for tick-borne encephalitis (TBE) infection.

Comparison of the Interferon γ-Binding Proteins of the Variola and Monkeypox Viruses

PCR was used to amplify DNA fragments containing the genes for interferon γ (IFNγ)-binding proteins (IFNγBPs) of the variola virus (VARV) and monkeypox virus (MPXV). The genes were expressed from baculovirus DNAs in Sf21 insect cells. The recombinant proteins were isolated from the culture medium by affinity chromatography. PAGE and Western blot analysis of the culture media and affinity-purified recombinant proteins showed that, in contrast to their cell analogs, the viral IFNγBPs form dimers in the absence of the ligand, human IFNγ. The biological activity of recombinant IFNγBPs was inferred from suppression of the protective effect of human IFNγ on L68 cells infected with the mouse encephalomyocarditis virus. The viral proteins showed a dose-dependent IFNγ-neutralizing effect. The prospects of using IFNγBPs as IFNγ antagonists are discussed.

3D structure modeling of complexes formed by CrmB TNF-binding proteins of Variola and cowpox viruses with murine and human TNFs

Orthopoxviral genomes bear genes for a series of homologous secreted proteins binding tumor necrosis factor (TNF). Orthopoxvirus species have different sets of these proteins. Variola virus has only one protein of this series, CrmB. Although CrmB protein sequences are similar to each other, their physicochemical and biological properties show certain species-specific features. We constructed 3D models of complexes formed by TNF-binding domains of variola and cowpox viruses with murine and human TNFs. We also constructed corresponding models with a mutant human TNF. In this mutant TNF, the arginine residue at position 31 involved in receptor binding was replaced by glutamine, characteristic of murine TNF. Analysis of the models showed that the least stable complex should be that formed by cowpox virus CrmB with human TNF, and the Arg31/Gln substitution should significantly stabilize the interaction between cowpox CrmB and mutant human TNF. Experimental comparison of the abilities of recombinant variola and cowpox CrmB proteins to inhibit the cytotoxic action of TNFs confirmed the predictions.

Visualization of CombiHIVvac Vaccine Particles Using Electron Microscopy

Acandidate vaccine CombiHIVvac is developed; presently the clinical phase I trial has been completed successfully. CombiHIVvac combines the conserved polyepitope immunogens approaches in a novel self-adjuvanted microparticle concept. The artificial TBI (T cell and B cell immunogen) polypeptide used in the vaccine comprises epitopes from Env and Gag. The polypeptide is conjugated to dextran and mixed with DNA, which leads to formation of microparticles presenting TBI on the surface and containing the DNA inside. The DNA (pcDNA-TCI) enclosed in the microparticles codes for the TCI (T cell immunogen) polypeptide, which contains CD8 and CD4 epitopes from Env, Gag, Pol, and Nef conserved among HIV subtypes A, B, and C. The proposed technique enables the vaccine components to combine into particles on the principle of self-assembly (Fig. 1A). The conjugate molecule consists of 1 dextran molecule, 1 protein TBI molecule, and 10–15 spermidine molecules. Positively charged spermidine provides binding of the conjugate dextran/protein TBI with negatively charged DNAvaccine promoting formation of particles on the self-assembly principle. According to our estimation, the plasmid pcDNA-TCI (6,583 bp) is able to present about 100–110 dextran (60 kDa) molecules on its surfaces. For vaccine assembly, pcDNA-TCI was added to conjugate in the proportion of 1 DNA molecule:120 conjugate molecules. To prove that CombiHIVvac has actually the form of particles, we used gel filtration chromatography and atomic force microscopy. During sepharose CL-2B gel filtration, the vaccine was eluted in the volume, corresponding to 12–14 MDa size material. Experimental visualization of a theoretically predicted formation of artificial microparticles was performed with transmission electron microscopy with negative staining (1%

Experimental Molecular Design of Combined Vaccines

A method was elaborated to construct combined artificial immunogens simulating virus particles. The gist was exposing protein antigenic determinants of one virus on the particle surface and delivering plasmids with genes for antigenic proteins of another virus to specialized immune cells. Such immunogens were constructed and shown to induce biosynthesis of specific antibodies against HIV-1 and the tick-borne encephalitis virus. The level and duration of the humoral and cell responses were assayed.