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Orthopoxviruses bear in their genomes several genes coding for homologous secreted proteins able to bind tumor necrosis factor. Different species of the genus possess different sets of these tumor necrosis factor-binding proteins. Viriola virus encodes the only one of them named CrmB. Despite sharing high sequence identity, CrmB proteins belonging to distinct orthopoxviral species were shown to significantly differ by their physico-chemical and biological properties. We modeled spatial structures of tumor necrosis factor receptor domains of variola and cowpox virus CrmB proteins bound to either murine, or human or mutated human tumor necrosis factor. In the sequence of last the arginine residue at position 31 is substituted with glutamine that is characteristic for murine tumor necrosis factor. Theoretical analysis of modeled ligand-receptor complexes revealed that the least stable should be the complex of cowpox virus CrmB with human tumor necrosis factor, and that arginine to glutamine substitution at position 31 should significantly stabilize binding of corresponding human tumor necrosis factor mutant to cowpox virus CrmB. Experimental evaluation of recombinant variola and cowpox virus CrmB efficiencies in inhibiting cytotoxic effect of all these tumor necrosis factors have approved our predictions.

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.

The phenotype of the dendritic cells (DC) generated from the adhesion fraction of mononuclear cells in the presence of GM-CSF and alpha-interferon was studied in patients with pulmonary tuberculosis. Despite the absence of significant differences in the count of mature CD83+DCs in the groups of patients (n = 38) and healthy donors (n = 30), elevated CD14(+)-monocyte levels and few activated CD25(+)-DCs were indicative of the impaired process of DC maturation/generation in patients with pulmonary tuberculosis, particularly in a subgroup of patients with a low T-cell proliferative response against PPD (PPD-anergy, n = 10). The patients with tuberculosis showed the lower relative levels of CD11c(-)-CD123(+)-DC and the normal levels of myeloid CD11c(+)D123(-)DCs. However, in patients with PPD-anergy, the content of myeloid CD11c(+)CD123(-)-DCs was significantly higher than that in PPD-reactive patients. Moreover, the patients with PPD-anergy were characterized by the elevated peripheral blood levels of CD14+CD16(+)-monocytes, which was associated with the high suppressive activity of monocytes (r(s) = 0.53; p < 0.05). The impaired process of DC generation/maturation in patients with pulmonary tuberculosis is believed to be associated with the changes in the phenotypic and functional properties of monocytes and to be a cause of an inadequate antigen-specific response in tuberculous infection.

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.

An original method for making effective artificial vaccines has been developed. Immunogens are virus-like particles containing a DNA molecule covered with recombinant proteins carrying the pathogen epitopes. The recombinant proteins are exposed on the surface of the particle and are attached to DNA via spermidine-polygluquine-glutathione or galactopyranoside conjugates (depending on the hybrid protein composition). Two variants of artificial immunogens-candidates for antiHIV vaccine have been prepared.

Evidence was obtained that the changed gravity, tension profiles of the magnetic fields inside the orbital station and other spaceflight factors (SFF) substantially influence the cell genome and synthesis of recombinant proteins. The authors proposed a technique of fixation of possible alterations in genes and expressed recombinant proteins in strains-producers of human alpha-interferon: HuIFN-alpha 2b, HuIFN-alpha 8a, HuIFN-alpha 10a, and HuIFN-alpha 14a. Spaceflight factors were simulated by way of gamma-irradiation at 10 Gy and 50 Gy by a cobalt unit, and centrifugation of samples at 10 g and 50 g. Cultivation of the strains-producers during the SFF simulation yielded HuIFN-alpha proteins with altered functional characteristics. Following exposure to simulated SFF, strains-producers expressed HuIFN-alpha that preserved a high titre of antiviral activity (AVA) but suppressed the antiproliferative activity (APA) inferring some structural/functional shifts in the HuIFN-alpha molecule due to, presumably, mutations in the active center responsible for APA. Determination of species specificity of the HuIFN-alpha recombinant proteins following exposure to SFF revealed dissociation of cross-AVA in homologous and heterologous cell cultures which can be also attributed to the structural/functional shifts in the HuIFN-alpha molecule. These changes can be localized in the receptor cluster of molecule or consequent to modification of the center defining HuIFN-alpha species specificity. The proposed simulation system allows fixation of shifts in the HuIFN-alpha structural/functional characteristics and investigations of the stability of eucaryotic genes in long-term space flights.

To solve the problems of biological safety of the cosmonaut on long-duration space mission and prediction of changes in macroorganism as a whole and constituting protein molecules under these conditions, it is important to study the influence of spaceflight factors (SFF) on microorganisms-carriers of modeled structures, protein molecules and pro- and eukaryotic genes in particular. Within the framework of scientific cooperation NAUKA-NASA, the authors proposed a model system of prokaryotic producers of pro- and eukaryotic proteins--staphylococcus alpha-toxin (SAT)--as a key protein in pathogenesis of staphylococcal infections, and human leukocytic interferon (HuIFN-alpha) as one of the homeostasis regulating proteins with well-studied structural and functional properties. Recombinant strains of E. coli with either a single or duplicated HuIFN-alpha 2b gene or other genes of the HuIFN-alpha family: HuIFN-alpha 8a, HuIFN-alpha 10a and HuIFN-alpha 14a were selected as producers of SAT and HuIFN-alpha. This biotechnologic system allows imitation and assessment of the SFF mutagenic effect both at the levels of genome of strain-carrier and gene-insertion and expressed CAT and HuIFN-alpha molecules including transcription, translation, assembly and post-translatory modifications of the target-protein. The developed methodology allows determination of highly mutable and conservative regions in the primary structure of a hypothetical protein associated with its functional activity, prediction of specific amino acid substitutes in these regions, and comparison of test calculations with a pool of natural mutations in the family of proteins under study. The structural/functional analysis of proteins and HuIFN-alpha genes made it possible to isolate and systematize functionally significant areas in the structure of hypothetical protein HuUFN-alpha, on the basis of which the most probable amino acid substitutions were prognosticated. This will present a possibility to identify expectable mutation events in HuIFN-alpha proteins, which so far have not been found in natural genes of the human interferon. Comparison of results of SFF modeling and space experiments aboard the International Space Station with monitoring of HuIFN-alpha mutant forms will help estimation of the extent of influence of the spaceflight factors on evolution of protein molecules.

Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

The nonstructural 36K protein of vaccinia virus (VV) mapped in HindIII-P and HindIII-J fragments of VV strain L-IVP has been expressed in E. coli as a fusion protein. The products of 36K gene preserved the antigen homology with the native protein 36K and the capacity to bind specific immunoglobulins of rabbit antiVV serum. The protective properties of 36K gene products and their joint effect with the immunodominant protein 35K were investigated. The non-structural 36K gene products showed no protective activity, but increased the production of specific antibodies in mice immunized with a mixture of both protein preparations, this increase being compatible with that observed after immunization with the inactivated virus preparations.

Using the oligonucleotide directed mutagenesis, a human lymphotoxin (TNF beta) mutant gene lacking 21 N-terminal codons has been obtained. Recombinant plasmid pLT21 for expression of the mutant gene has been constructed. The mutant gene in the plasmid was placed under control of a tandem of constitutive promoters from coliphage T7. A simple procedure for isolation of recombinant protein was developed. The procedure allows to obtain the highly purified biologically active mutant protein with a good yield. During biosynthesis the recombinant protein undergoes a posttranslational processing resulted in the cleavage of N-terminal methionine and leucine residues.

The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

Using a chemically synthesised adapter, the coding part of an artificial gene for human leukocyte alpha 2 interferon (ifn-alpha 2) has been duplicated. The adapter contained a termination signal of the first gene (TAA) within the Shine-Dalgarno sequence of the second gene (TAAGGA), distance between the terminating codon and starting codon of the second gene being 11 nucleotides. In another case this distance was 69 nucleotides, with the same SD sequence. The expression of the tandems as a part of polycistrons has been studied under control of promoters Plac, (Ptrp)2 of E. coli, and PVIII of M13 phage. It was found that tandems of ifn-alpha 2 genes in polycistronic structures trp L-ifn-ifn and IX-VIII-ifn-ifn under control of promoters (Ptrp)2 and PVIII, respectively, provided high level of the interferon biosynthesis, thus differing from the tandem under Plac promoter control, which had only ifn-ifn translation coupling.

Using gene fragments encoding the leader peptide of E. coli tryptophane operon (as duplicated fragment HhaI-140) or M13 phage coat protein (as TaqI-381 or HaeIII-1623 fragments) and basing on pDS1 family of plasmids, expression vectors have been constructed which contained transcription promoters Ptrp, PVIII, and Pv + PVIII, respectively. An artificial gene for human leukocyte interferon alpha 2 (ifn-alpha 2) has been cloned into these plasmids, so that its transcription was a part of polycistronic mRNA and preceding translation was terminated upstream to the ribosome binding site and starting codon of the interferon gene. E. coli cells harbouring these recombinant plasmids provided high level of the interferon biosynthesis. The effect of the mRNA length on the amount of protein synthesised under control of the M13 coat protein transcription-translation signals has been found.

In vitro and in vitro transcription of the region of lambda phage DNA containing promoter Patt has been studied. Active transcription was observed in the vicinity of the att site and at the promoter Patt in vivo during the first minutes after infection. This transcription considerably decreases at the 1th minute, and stops completely at the 15th minute after infection. Maximum synthesis of integrase also takes place at the 15th minute after infection. In vitro the partially purified int protein (integrase) completely inhibited transcription from the promoter Patt. Hence, integrase is most probably a repressor of Patt in vivo and in vitro.

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.

Plasmid expression vector using the temperature-regulated promoter P'R of bacteriophage lambda is described. The vector carries a combination of two regions of lambda cI857indgenome, that contain: 1) gene cI and promoter PR, and 2) gene Q and promoter P'R. Transcription or gene Q is initiated at promoter PR, which is controlled by cI857 repressor. The Q gene product acts as a positive regulator of RNA synthesis from P'R. At 37 degrees C, sufficient amounts of protein Q are synthesised to initiate the expression of the cloned gene from P'R. Inactivation of Q gene (by elimination of a single NcoI site) results in the loss of P'R expression activity in the vector. E. coli beta-galactosidase gene (lacZ) and human leukocyte interferon alpha 2 gene (ifn alpha 2) were cloned into a single EcoRI cleavage site under the control of P'R. These constructs express high levels of beta-galactosidase and interferon alpha 2 in E. coli at 37 degrees C.

A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence. Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription. It was found that efficiency of beta-galactosidase synthesis in E. coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end. High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position. Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon.

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.