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The chicken anemia virus gene that encodes apoptin, a selective killer of cancer cells, was synthe-sized and inserted into the vaccinia virus (strain L-IVP) genome. The insertion replaces a major part of the viral C11R gene that encodes viral growth factor, which is important for virulence. The recombinant virus VVdGF-ApoS24/2 was obtained by transient dominant selection using the gene of puromycin resistance as a selective marker. The expression of the apoptin gene from a synthetic early-late promoter of vaccinia virus ensured the efficient accumulation of the target protein in VVdGF-ApoS24/2-infected cells. Although recombinant apoptin carried the signal peptide of the virus growth factor at the N-end, the protein was not secreted into the culture medium. The recombinant virus VVdGF-ApoS24/2 exhibited significantly higher selective lytic activity in human cancer cell lines (A549, A431, U87MG, RD, and MCF7) than the parent strain L-IVP and its VVdGF2/6 variant with C11R deletion. These results suggest that the use of apoptin can be an efficient means of enhancing the natural anticancer activity of vaccinia virus.

Comparative analysis of amino acid and nucleotide sequences of OTF variable terminal segments in the genome from numerous variola virus (VARV) strains has detected a locus for viral genotyping according to the geographic region, i.e., the OTF O1L region of VARV. The primers for the synthesis of the OTF fragment by the polymerase chain reaction (PCR) were calculated in order to distinguish VARV South-American and West-African genotypes from other virus strains. Asian and African (except West African) strains have been reliably differentiated by PCR-RFLP analysis. This method has been tested on 16 VARV strains from various geographic regions. The developed approach is simple, fast, and reliable for further applications.

Two segments of the variable terminal regions of the variola virus (VARV) genome were sequenced in 22 strains from the Russian collection, including about 13.5 kb of the left segment and about 10.5 kb of the right segment. The total length of the sequences was over 540 kb. Phylogenetic analysis of the new and published data determined the relationships among 70 VARV strains, the character of their clustering, and the intergroup and intragroup variation of the strain clusters. Loci with the highest polymorphism rate were identified and proved to map to noncoding regions or to regions of damaged open reading frames, characteristic of the ancestral virus. These loci offer attractive possibilities for developing a strategy of VARV strain genotyping. Recombination analysis by different methods did not detect, except for a single case, significant recombination events in the VARV strains examined.

Comparative RFLP analysis was for the first time performed for 21 variola virus (VARV) strains of the Russian collection with 20 amplicons covering the total VARV genome. The amplicons were synthesized in the long polymerase chain reaction. A database useful as a reference for identifying VARV strains was generated. VARV strains isolated in different geographical regions were compared and proved to vary mostly in variable genome regions. Each of the dendrograms constructed included three clusters of African, Asian, and VARV-alastrim isolates. The VARV-alastrim isolates differed to the greatest extent from the other strains. VARV strains isolated during an ecdemic variola burst in Moscow (1960) grouped with Asian isolates. Polymorphism of VARV strains was for the first time observed for a single variola burst with a few affected patients.

Two systems have been examined for delivery of DNA-vaccine encoding a HIV-1 polyepitope CTL-immunogen (TCI). One is intended for i.m. injection and is in the form of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine–polyglucin conjugate. The other is intended for mucosal immunization and is based on attenuated Salmonella typhimurium strain 7207, which can deliver pcDNA-TCI directly into professional antigen-presenting cells (APC). After immunization, the artificial VLP and recombinant Salmonella induced an enhanced HIV specific serum antibody, proliferative and CTL responses compared to those induced by naked pcDNA-TCI. The most significant responses were produced when pcDNA-TCI was delivered by Salmonella. © 2004 Published by Elsevier Ltd.

Integrative plasmids pΔC, pΔD, and pΔG were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chick embryos.

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-α and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-κB, STAT3, PI3K, and β-catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.

To study specific properties of the human gamma-interferon (gamma-IFN) receptor-like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E. coli cells have been constructed. The recombinant viral gamma-IFN receptor-like proteins have been found to have high interferon-neutralising activity with regard to human gamma-IFN but not murine gamma-IFN and human alpha-IFN. The variola major and variola minor proteins under study do not differ in the efficiency of human gamma-IFN antiviral activity inhibition.

Potential ORFs were sought in the extended segments of terminal variable regions in the variola virus genome. These ORFs underwent a detailed structural and functional analysis and were compared both between themselves and with homologous ORFs of various orthopoxviruses. The most conservative and heterogeneous ORFs of 70 VARV strains were detected. The unique for VARV ORF (111 a.a) was revealed for Helder and Mary strains of the Russian collection. In addition, only in the Helder strain, ORF D14L was disintegrated into two separate ORFs. A number of ambiguities were found in the current databases for VARV ORF. The dominating type of evolution was ascertained to be stabilizing selection for analyzed ORF. It has been established that VARV ORF C3L, unlike other poxviral orthologs, undergoes an adaptive selection. These findings suggest that this gene plays an important role in human VARV adaptation.

Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8 + CTL) and T-helper lymphocytes (CD4 + Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database . The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

A synthetic gene for human angiogenin was cloned into pRTang under control of the Proteus mirabilis recA promoter. After induction with mitomycin C, Escherichia coli cells transformed with pRTang produced an additional 14.2-kDa protein. According to electrophoretic and immunochemical analyses, this protein was identical to human angiogenin.

Chemical-enzymatic synthesis and cloning of a gene for a human anaphylatoxin C5a analog were carried out. Recombinant plasmid pRC5a providing the expression of the synthetic gene in the Escherichia coli cells was obtained. The biological activity of the expression product was demonstrated by the chemotaxis activity test and by the release of myeloperoxidases from rat peritoneal cells that was induced by bacterial cell lysates containing the recombinant protein.

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.

Sharpening of frequency selectivity of the mechanical part of the hearing system due to the transformation of the signals in the receptor system is investigated. It is proposed that excitation of neuron endings is proportional to the amplitude of the current which was produced by the difference of microphonic potentials in the neighbouring hair cells. It is shown that possible degree of the selectivity sharpening of the cochlea which can be explained by this model is in a good agreement with the known experimental results.