[Recombinant full-size human antibody to Ebola virus].

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.

[Experimental evaluation of the protective properties of some nonwoven materials for respiratory protection means and prospects of their use].

The protective properties of nonwoven materials (Spandbond, SMS) used to manufacture 3-5-layer medical masks, by using model physical and bacterial test aerosols, were experimentally assessed. It was shown that the more layers of the materials, the less permeable they became for test aerosols. Three-five-layer masks made from SMS at a density of 42 g/m2 were found to have higher protective properties for oil mist and fine aerosol than those made from Spandbond at a density of 25 g/m2. Five-layer SMS materials at a density of 42 g/m2 have the highest values of bacterial aerosol retention.

[Clinical trials of oral recombinant bivaccine against variola and hepatitis B during double vaccination].

Clinical trials of oral live recombinant embryonic variola and hepatitis B bivaccine as tablets (Revax-BT) were performed. When volunteers were prevaccinated with oral variola vaccine first in a small dose and, 7, 14, 30, 90, and 180 days later, in a larger dose, a slight reactoginicity was sometimes observed after the first vaccination (with a small dose) whereas revaccination with a larger dose did not give rise to any clinical manifestations. A month after vaccination, a protective level of virus-neutralizing antibodies to vaccinia virus (VV) was observed in 90-100% of the volunteers twice immunized with the bivaccine (in a small dose and in a larger one at an administration intervals of 1-2 weeks under remote revaccination while 6-9 months following vaccination, this level was recorded in 80% of the volunteers. A month following vaccination, 50-55% seroconversion to VV was observed in the volunteers twice immunized with the bivaccine (at an interval of 1 or 3-6 months). Cellular immunity to VV was low (0-20%). Double immunization of volunteers with the oral bivaccine under remote vaccination failed to produce the significant levels of humoral and cellular immune responses to hepatitis B markers. Recombinant VV was not recorded in any blood, saliva, and urine samples taken in the volunteers twice immunized with the bivaccine.

[Evaluation of the protective properties of nonwoven materials for throw-away medical overalls].

The permeability of varying density felts (Spandbond, SMS, Tyvek, Sontara) for throw-away medical overalls was experimentally assessed under statistic and dynamic conditions, by using model test aerosols, including bacterial one. Their permeability was shown to decrease with the higher density of the materials under study. Laminated Span-bond contributes to an abrupt reduce in the bacterial penetration of this kind of tissues and enhances their protective properties. The felts CMC, Tyvek, Sontara, and laminated Spanbond were found to have the highest protective properties. In terms of their barrier and protective properties, the felts surpass the tissues used to make re-usable protective medical overalls.

[Human recombinant antibodies to variola virus].

Eight specific antibodies to live variola virus (VV), Ind-3a strain, and 7 antibodies to VV, Butler strain, were selected from the synthetic combinatorial phage display library on single-chain (scFv) human antibodies. Indirect solid-phase enzyme immunoassay showed the ability of these antibodies to bind the VV strains Ind-3a, Butler, Brazil-131, Kuw-5, and Congo-2. Moreover, earlier selected human scFv antibodies were also tested in the reaction of binding to the above VV strains. The experiments could reveal the antibodies that bound alastrim strains more effectively that did other VV strains. The nucleotide sequences encoding for the selected scFv antibodies were determined.

Ankyrin-like proteins of variola and vaccinia viruses.

Computer analysis of full coding sequences of variola major virus strain India-1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin-like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.

Genes of variola and vaccinia viruses necessary to overcome the host protective mechanisms.

Analysis of variola virus nucleotide sequence revealed proteins belonging to several families which provide the virus with the possibility of overcoming the barriers of specific and non-specific host defence against viral infection. The complement-binding proteins, lymphokine-binding proteins, and serine protease inhibitors can be assigned to this type, as can the proteins providing the orthopoxviruses with resistance to interferon. The revealed differences between the genes (proteins) of variola and vaccinia viruses under study are discussed.

Comparison of the genetic maps of variola and vaccinia viruses.

The complete genetic map of the variola major virus strain India-1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Analysis of the nucleotide sequence of 23.8 kbp from the left terminus of the genome of variola major virus strain India-1967.

Sequencing and computer analysis of the nucleotide sequence of variola major virus strain India-1967 (VAR-IND) DNA segment (23 786 bp) covering the left variable region of the viral genome has been carried out. Twenty-nine potential open reading frames were identified. Structure-function organization of the VAR-IND DNA segment was compared with previously reported sequences from analogous genome regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR). Multiple structural differences between the VAR-IND and genome regions were analysed and both VAC-COP and VAC-WR have been found. Possible molecular factors of virulence, virus host range genes as well as differences revealed in the structure of these genes of VAR and VAC will be discussed.

Analysis of the nucleotide sequence of a 43 kbp segment of the genome of variola virus India-1967 strain.

Sequencing and computer analysis of the nucleotide sequence of the variola virus strain India-1967 (VAR) genome segment (43069 bp) from the region of HindIII C, E, R, Q, K, H DNA fragments has been carried out. Forty-three potential open reading frames (ORFs) have been identified, and the polypeptides encoded by them have been compared with the analogous proteins of vaccinia virus strain Copenhagen (COP). ORF E7R of VAR is much shorter than the COP analog. The other polypeptides coded by the potential ORFs of VAR are highly conserved in comparison with COP. Possible functions of the predicted viral polypeptides are discussed.

Analysis of the nucleotide sequence of 48 kbp of the variola major virus strain India-1967 located on the right terminus of the conservative genome region.

Computer analysis of a variola major virus (VAR) genomic fragment bounded by the open reading frames (ORFs) D1R and A33L, which is 47,961 bp long, revealed 46 potential ORFs. The VAR proteins were compared to the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA-capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion bodies protein. Possible functions of the analysed viral proteins are discussed.

Analysis of the nucleotide sequence of 53 kbp from the right terminus of the genome of variola major virus strain India-1967.

Sequencing and computer analysis of a variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region has been carried out. Fifty-nine potential open reading frames (ORFs) of over 60 amino acid residues were identified. Structure-function organization of the VAR-IND DNA segment was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple differences between VAR-IND and the strains of VAC but the high identity of VAR-IND with VAR-HAR in the genetic maps are revealed. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

Immunization with recombinant vaccinia viruses expressing structural and part of the nonstructural region of tick-borne encephalitis virus cDNA protect mice against lethal encephalitis.

Three recombinant vaccinia viruses containing different fragments of tick-borne encephalitis virus (TBEV) cDNA representing the 5'-noncoding region (5'NCR), all structural and part of the nonstructural regions were constructed. Western blot analysis showed that E and NS1 proteins were expressed and processed correctly in cells infected with recombinant viruses vC-NS1 (coding for C-prM-E-NS1 region) and vC-NS3 (coding for C-prM-E-NS1-NS2A-NS2B-NS3 region). In contrast, in cells infected with recombinant virus v5'C-NS2A (coding for 5'NCR and C-prM-E-NS1-NS2A regions) expression of NS1 protein was greatly reduced and no E protein was detected. Immunization of mice with vC-NS3 induced high levels of TBEV-specific antibodies and protected them against intraperitoneal challenge with 10(7) LD50 of TBEV. The level of protection was very similar to the level of protection achieved by immunization with commercially available inactivated TBEV vaccine. Although the immunization of mice with recombinants vC-NS1 and v5'C-NS2A induced much lower levels of TBEV-specific antibodies, they were still protected against intraperitoneal challenge with 10(4) and 10(3.6) LD50 of TBEV, respectively. The high level of protection against TBEV infection achieved by the immunization of mice with the recombinant vaccinia virus vC-NS3 makes this virus a very attractive candidate for development of a live recombinant vaccine against TBEV.

Design of immunogens as components of a new generation of molecular vaccines.

Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.

[Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea]. / Vnutrikletochnyi transport ribosomnoi RNK iadernogo proiskhozhdeniia u Acetabularia mediterranea.

The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.

[Intracellular distribution of RNA in Acetabularia mediterranea]. / Izuchenie vnutrikletochnogo raspredeleniia RNK u Acetabularia mediterranea

RNA synthesis and distribution in cytoplasm of Acetabularia mediterranea cell were studied by radiochemical and ultramicrobiochemical methods. Existance of an apico-basal gradient of RNA concentration in the cytoplasm was shown. Variations in the rate of the RNA synthesis in different regions of the cytoplasm are of great importance for the formation of this gradient whereas a contribution of nuclear RNA transport is insignificant. Sedimentation coefficients of about 23S and 16S were detected for the most part of the synthesized cytoplasmic RNAs. Evidently these RNAs represent rRNA of chloroplast ribosomes. Basal region of cytoplasm contains a relatively large amount of low molecular weight RNA. The differences in stability of newly-synthesized RNA were found in cytoplasmic regions under examination.

[Comparative restriction enzyme analysis of the genome in variola virus strains from the Russian collection]. / Sravnitel'nyi restriktsionnyi analiz genomov shtammov virusa natural'noi ospy iz Rossiiskoi kollektsii.

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.