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Efficacy of candidate DNA-vaccines based on the variola virus natural gene A30L and artificial gene A30Lopt with modified codon usage, optimized for expression in mammalian cells, was tested. The groups of mice were intracutaneously immunized three times with three-week intervals with candidate DNA-vaccines: pcDNA_A30L or pcDNA_A30Lopt, and in three weeks after the last immunization all mice in the groups were intraperitoneally infected by the ectromelia virus K1 strain in 10 LD50 dose for the estimation of protection. It was shown that the DNA-vaccines based on natural gene A30L and codon-optimized gene A30Lopt elicited virus, thereby neutralizing the antibody response and protected mice from lethal intraperitoneal challenge with the ectromelia virus with lack of statistically significant difference.

Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

There are numerous viral proteins known to date to modulate protective responses of their hosts. Representatives of the Poxviridae family have the greatest number of genes coding for proteins, inhibiting inflammatory responses, activities of interferons, regulating immune reactions and other protective mechanisms of macroorganisms, among viruses. This review regards poxviral immunomodulatory proteins--namely, complement-binding proteins, inhibitors of serine proteases, chemokine- and TNF-binding proteins --that were shown to be efficient therapeutics in various animal models of inflammatory and autoimmune diseases. The prospects of their usage in clinical practice for treating human inflammatory and autoimmune disorders are discussed.

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

Poxviruses, belonging to the genus of Orthopoxviruses and Leporipoxviruses, express a protein that binds a wide spectrum of CC-chenokines. The above viral protein (vCCI) has no homology by the amino acid sequence with the known cellular receptors of chemokines or with any other proteins represented in databases. Sixty-nine nucleotide sequences of the vCCI protein gene of 4 Orthopoxviruses, which are pathogenic to man, and of 2 species, to whom man is immune, as well as 16 of such sequences borrowed from GenBank were subjected to the phylogenetic analysis. Strains of Orthopoxviruses, except for cowpox viruses (CPXV), were found to cluster together in groups according to their specific attribute. CPXV isolates can be conditionally divided into 2 subgroups, which are much remote from one another evolutionally. Four subgroups were defined for smallpox virus with their strains originating from different geographic regions.

The review considers recent data on the structural-functional organization of the genome of orthopoxviruses pathogenic for humans, including the variola, monkeypox, cowpox, and vaccinia viruses. Emphasis was placed on the structure of molecular virulence factors that suppress the inflammatory reactions, immune response, and interferon effects induced by virus infection.

This is a critical review of the modern methods of gene mapping in the vaccinia virus genome. Experimental data are given on the localization of the vaccinia virus genes. Major attention is paid to the functions of the proteins encoded by the mapped genes.

A hybrid vaccinia virus expressing a chimeric protein consisting of thymidine kinase and the encephalitogenic determinant, S1, from guinea pig myelin basic protein was constructed. Infection of guinea pigs with the virus resulted in the development of allergic encephalomyelitis.

Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.

Vaccinia virus gene encoding 36K protein was cloned in pUR290 bacterial expressing vector and resulted in the synthesis of a chimeric protein in E. coli. The chimeric protein consists of beta-galactosidase and virus protein in C-termini. It has virus antigen specificity. By monospecific antibody 36K protein of vaccinia virus was determined to be non-virion. It is localized in the cytoplasm of infected cells.