[Autoimmune response in HIV-infected patients is directed against CD4 domain 4].

Phage display epitope library technology and a novel computer algorithm have been used for the localization of CD4 epitopes specific for monoclonal antibody (mAb) T6 and autoimmune antibodies found in an HIV infected patient. Both predicted epitope clusters have been shown to overlap and to be localized within the domain 4 of CD4. They included Cys303, Glu304, Glu330, and Lys331 amino acids. Few amino acids predicted by the algorithm as the epitope residues and two residues that did not relate to the epitope were sequentially substituted for Ala. Further analysis of the mutated forms of sCD4 expressed in 293T cells transfected with the corresponding DNAs, supported the predicted localization of the mAb T6 epitope. The results demonstrate that the autoimmune response in HIV-infected patients is directed against domain D4 of sCD4.

Dependence of conformation of D3/D4 domains of human CD4 on glycosylation and membrane attachment.

Conformational dynamics of human T-helper cell receptor protein CD4 has been studied with the help of monoclonal antibody (mAb) T6. The mAb T6 discriminates between s- and m-forms of CD4 and recognizes a specific conformation of the soluble (s) form of CD4 including the first nine amino acids of CD4 transmembrane sequence. However, change of tryptophan for serine in position 2 in this sequence destabilizes the T6-type conformation. By enzymatic deglycosylation and deletions of glycosylation sites, we show that T6-type conformation depends on glycosylation in both sites (Asn271 and Asn300). We show also that the sugars are not involved in direct binding to the antibody but stabilize the D3/D4 local conformation. Deglycosylated forms of sCD4 in vivo acquire a specific conformation similar to the wild type sCD4, which however cannot be restored after denaturation/renaturation under conditions of non-reducing Western blot. This observation indicates that the correct protein folding needs chaperone assistance and cannot be achieved in vitro. Completely non-glycosylated sCD4 is synthesized and secreted into the growth medium. In the medium, this mutant appears to be unstable and aggregates during time. In a contrast to soluble CD4, mutations in glycosylation sites abrogate expression of membrane CD4, thus demonstrating a different secretion pathways for soluble and membrane proteins.

[Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

[Anti-CD4 autoimmunity in HIV-infected persons in Russia].

The frequency of anti-CD4 antibodies was determined in the sera or plasma derived from the patients infected with HIV-1 belonging to different genetic subgroups. The anti-CD4-antibodies in a dilution of > or = 1:1000 were found in 14% of the patients infected with the gagA/envA virus characteristic for injectable drug users in East Europe. The frequency of autoimmune antibodies among the HIV-infected patients with envB virus was substantially less (4.4%). Competitive ELISA using monoclonal antibodies to different CD4 domains demonstrated that irrespective of the viral genotype, the autoimmune epitope is located within the D4 or D3/D4 domains of CD4 receptor.

[Synthesis of recombinant proteins from the American and European viruses of the porcine reproductive and respiratory syndrome in baculovirus expression system and their antigenic properties]. / Sintez v bakulovirusnoi sisteme rekombinantnykh belkov amerikanskogo i evropeiskogo tipov virusa reproduktivnogo i respiratornogo sindroma svinei i ikh antigennye svoistva.

The baculovirus expression system was made use of to derive the recombinant nucleocapsid (N) proteins of the American and European virus types and of the porcine reproductive and respiratory syndrome (PRRS). The obtained products were purified by metal-affine chromatography and their specificity was confirmed in immunechemical reactions with reference monoclonal antibodies. The antigenic activity of recombinant proteins was studied by indirect immune enzyme assay (IEA) with porcine serum, which had been in advance characterized by the "HerdCheck" kit (IDEXX Co.). It was shown as possible to apply the derived recombinant antigens in determining, by indirect IEA, antibodies to the PRRS virus.

[Synthesis and immunochemical properties of the recombinant major surface glycoprotein E2 of the classical swine fever virus]. / Sintez i immunokhimicheskie svoistva rekombinantnogo glavnogo poverkhnostnogo glikoproteina E2 virusa klassicheskoi chumy svinei.

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.

[Inhibition of nuclear import of HIV-1 Vpr protein by Gag polyprotein in the process of virus-like particles formation]. / Ingibirovanie iadernogo importa belka VPR VICh-1 poliproteinom GAG v protsesse formirovaniia virusopodobnykh chastits.

For studies of intracellular Vpr transport and the effect of the HIV-1 Gag polyprotein on the process, a recombinant baculovirus strain was constructed, which directs the synthesis of Vpr fused with the baculovirus secretory polypeptide. During infection the majority of Vpr has been observed in the cell nuclear fraction. These data suggest that Vpr nucleophilic signal is more active than the secretory one. However, during Vpr and Gag co-expression in the baculovirus expression system, Vpr content in the nuclei is decreased, since this protein incorporates effectively into virus-like particles and forms stable complexes with Gag polyprotein. Presumably, the Vpr-Gag post-translational interactions are needed for the Vpr incorporation into virions and suppress the nuclear import of this protein.

[Interaction of gag polyprotein-precursors p55 and p48 with p160gag-pol during formation of virus-like particles of HIV-1 with recombinant strains of vaccinia virus]. / Vzaimodeistvie gag poliproteinov-predshestvennikov p55 i p48 s p160gag-pol pri formirovanii virusopodobnykh chastits VICh-1 rekombinantnymi shtammami virusa ospovaktsiny.

The polypeptide composition of HIV-I virus-like particles produced by CV-I cells during mono- and coinfection with recombinant vaccinia virus (rVV) strains containing the whole (p55) and carboxyterminal truncated (p48) gag genes and gag-pol sequence is studied. In monoinfection both the gag-strains actively produced virus-like particles consisting of non-processed p55Gag and p48Gag polyprotein without p6 domain. In case of a coinfection of the cells with one of these strains and the rVV producing p160Gag-Pol polyprotein the virus-like particles consisted of p24 protein and a negligible amount of non-processed Gag precursors. The share of p24 protein increased in proportion to the duration of coinfection and decreased with a reduction of multiplicity of infection with rVV carrying p160Gag-Pol. Hence, the absence of p6 domain does not influence the processing of Gag proteins during virus-like particles assembly and budding. In contrast to the natural systems of HIV-I development, in the rVV expression system the p6Gag domain virtually does not contribute to reactions between Gag and Gag-Pol precursors and to the particles' morphogenesis.

[Expression of human immunodeficiency virus gag antigens and formation of virus-like particles in a cell culture, infected with recombinant vaccinia virus strains (an electron-microscopic study)]. / Ekspressiia gag-antigenov virusa immunodefitsita cheloveka i obrazovanie virusopodobnykh chastits v kul'ture kletok, infitsirovannykh rekombinantnymi shtammami virusa ospovaktsiny (élektronno-mikroskopicheskoe issldovanie).

The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied. The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29). The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.

[Expression of the vif gene of human immunodeficiency virus type 1 in Escherichia coli and study of the immunoreactivity of the vif protein]. / Ekspressiia gena vif virusa immunodefitsita cheloveka tipa 1 v kletkakhEscherichia coli i issledovanie immunoreaktivnosti vif-belka.

Full-sized gen vif of human immunodeficiency virus has been synthesized and cloned into plasmid pGEX-2T. Vif-gene expression was found in Escherichia coli cells resulting in production of a hybrid GST-protein. The recombinant protein studied by the immunoblotting technique reacted with 8 of 22 probes of human HIV-positive sera. The recombinant protein is specifically cut by thrombin in two proteins corresponding to GST and VIF-proteins in molecular mass.

[The antigenic and immunogenic properties of a recombinant protein of the HIV-1 gag-pol gene secreted by Saccharomyces cerevisiae yeast cells]. / Antigennye i immunogennye svoistva rekombinantnogo belka gena gag-pol VICh-1, sekretiruemogo drozhzhevymi kletkami Saccharomyces cerevisiae.

Analysis of the immunological properties of recombinant proteins of HIV-1 gene gag-pol secreted by yeast cells S. cerevisiae was carried out. The proteins under study interacted with antibodies from HIV-1-seropositive human subjects and with antibodies of rabbit immune serum to the native virus as effectively and specifically as natural HIV-1 proteins. The yeast gag-pol-protein complex was markedly immunogenic and induced in animals synthesis of antibodies of a certain specificity spectrum. A comparative immunochemical analysis of the properties of the recombinant proteins carried out by EIA and immune blot showed a certain degree of similarity between the yeast proteins and those of analogous construction produced in E. coli system.

[The immunoenzyme test system for detection of HIV-1 antigens based on using immune polyclonal anti-HIV serum and monoclonal antibodies against gene GAG HIV-1 proteins]. / Immunofermentnaia test-sistema dlia vyiavleniia antigenov VICh-1, osnovannaia na primenenii immunnoi poliklonal'noi anti-VICh syvorotki i monoklonal'nykh antitel k belkam gena GAG VICh-1.

The paper describes the enzyme immunoassay system for detection of human immunodeficiency virus antigens, which is based on the use of rabbit anti-HIV antibodies and monoclonal antibodies to HIV-1 gene proteins gag. The system may be useful in the examination of laboratory and clinical samples to reveal both free and conjugated antigens in the composition of immune complexes. The sensitivity of the assay system under development is 0.5 ng/ml at 100% specificity.

[Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]. / Poluchenie immunofermentnykh kon"iugatov beta-laktamazy iz Bacillus licheniformis 749/c i peroksidazy khrena s chelovecheskimi antitelami k VICh-1.

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.

[Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens]. / Metody polucheniia i selektsii rekombinantov virusov ospovaktsiny, ékspressiruiushchikh chuzherodnye virusnye antigeny.

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.

[Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin]. / Konstruirovanie rekombinantnogo shtamma virusa ospovaktsiny, soderzhashchego ékspressiruemyi gen gemaggliutinina virusa grippa A.

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.

The nucleotide sequence of the leftmost XhoI fragment (6%) of simian adenovirus SA7P.

The DNA of simian adenovirus SA7P was cloned in pBR322. The nucleotide sequences of the leftmost 2238 bp and the rightmost 188 bp of the viral genome were determined. SA7P DNA has an inverted terminal repeat of 183 bp. The sequence at the left terminus exhibits extensive homology with that of the E1 regions of human adenovirus 5, 7 and 12 DNAs. Based on this homology, the RNA coordinates and coding regions could be deduced. The sequenced SA7P DNA contains the entire E1A and part of the E1B region.

[Isolation and characteristics of a mutant herpes simplex virus type 1 resistant to phosphonoacetic acid]. / Vydelenie i kharakteristika mutanta virusa gerpesa prostogo tipa 1, rezistentnogo k fosfonouksusnoi kislote.

By successive passages and triple cloning of herpes simplex virus type 1 (HSV-1) in Vero cell culture in the presence of increasing concentrations of phosphonoacetic acid (PAA) a mutant of HSV-1 resistant to PAA (PAAr) was derived and characterized. The resistance to the inhibitor was transmitted from PAAr-mutant to a sensitive strain (L2) by recombination performed by the marker rescue method using DNA fragmented by Hpa-1 restrictase. The resulting recombinant (R-551) was resistant to the inhibitor and had an altered primary structure of DNA.

[Resistance of various herpes simplex virus strains to the action of chemical inhibitors]. / Izuchenie tsoichivosti razlichnykh shtammov virusov gerpesa prostogo k deistviiu khimicheskikh ingibitorov.

The effect of chemical inhibitors on reproduction of 2 laboratory and 3 vaccine strains of herpes simplex virus (HSV), types 1 and 2, was studied. By the time of the study the vaccine strains had undergone from 27 to 69 passages in chick embryo fibroblast cultures. All the vaccine strains (L2, Us, and VN) exhibited 100-1000 fold higher resistance to phosphonoacetic acid than did the laboratory F+ and G strains, and the vaccine L2 strain (HSV-1) was also 1000-fold resistant to 1-beta-D-arabinofuranosine thymine.