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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.

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.

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.

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.

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.

The reactogenicity of the embryonic live recombinant variola and hepatitis B bivaccine as tablets (Revax-BT) as well as its safety and immunogenicity were evaluated in clinical trials made in volunteers who had previously immunized or not with variola vaccine. A preliminary conclusion was made on a lack of side effects and drug safety in primary vaccination and been revaccination with low and high doses. Primary immunization of volunteers and as bivaccination with high doses stimulated the most pronounced immune response to the vaccine virus versus such effect observed in immunization of volunteers with low vaccine doses. Humoral immune response to HBs was observed in 75% of volunteers of both groups after as bivaccination. Such response was most pronounced in examinees immunized with low vaccine doses versus those who received high bivaccine doses. At the same time, no protective levels of humoral immunity response to HBs Ag were observed in volunteers first vaccinated.

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.

A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.

Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (MCA) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.

A method for describing the Orthopoxviruses that are pathogenic both to man and animals is described in the article. The method is based on hybridization of a fluorescently labelled amplified DNA sample with oligonucleotides, which were immobilized in a microchip. Species-specific regions within the crmB gene encoding a viral analogue of the tumor necrosis factor receptor, i.e. an important gene determining the pathogenicity of the mentioned Orthopoxviruses type, were used as a target for identification. The identification procedure takes around 6 hours and does not demand any costly equipment (a portable fluorescent microscope can be used).

Elaboration of an anti-HIV vaccine is a highly important task because there is a need to arrest or at least to slow-down the rapid spread of AIDS throughout the world. Regrettably, no attempts to create an effective vaccine resulted in success. Nonetheless, the available data contribute to building up the confidence in that the set purpose can be achieved provided extra resources are found for working out a potential anti-HIV vaccine. The paper contains some results of research conducted by the "Vector" Research Center for Virology and Biotechnology in the field of artificial polyepitope immunogens, which could be potential anti-HIV vaccines, and in the field of creating various system for their delivery. The immunogenic properties of the thus obtained vaccine structures were tested on mice BALB/c. The delivery systems were experimentally demonstrated to ensure the induction of specific antibodies against HIV-1, with such anti-bodies having a virus-neutralizing activity; the above systems also induce the cellular immunity.

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Trh-297, Phe-383, Tyr-384, Arg-419, Ile-420, Thr-415, Leu-416, Pro-417, Lys-421, and Trp-112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

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 DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (Y waves phi) with a period of about 3 Mbp. Distribution of G + C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G + C than chromosome 22. Both exons and Alu repeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alu repeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alu repeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Alu distribution patterns along the chromosome, the concurrent distribution of Alu repeats in both orientations along the chromosome, and the equal copy numbers for Alu in direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alu repeats belong to parasitic (junk) DNA.

A phase peptide library was screened with virus-neutralizing monoclonal antibodies (MCA) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MCA 2F5 were selected by ELISA. Amino acid sequence analysis revealed a homology to region 662-671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in serum of immunized rabbits.

Specific biological activity of the domestic preparation of recombinant human erythropoietin (RHEPO) in an original pill form was experimentally studied in vitro and in vivo. Administration of the RHEPO parent substance at a concentration of 0.5 10 U/ml significantly accelerated the growth of erythroid colony-forming cells (CFU-E) in the culture of intact nonadherent myelokaryocytes. The erythropoiesis-stimulating properties of the new RHEPO preparation are comparable with those of the well-known injection preparations. The drug action is related to increasing the erythroid cell content in the bone marrow and the reticulocyte and erythrocyte counts in the peripheral blood.

The effects of ointment containing king crab (Paralithodes camtschatica) collagenase on intact skin, thermal, and pyonecrotic wounds were studied in rats by using hematological, biochemical, immunological, and morphological methods. The ointment for the skin and viscera was shown to be safe. It is highly effective in debriding the infected wounds. Different concentrations of collagenase were tested. The concentration of collagenase was recommended to be 0.2 mg/g ointment for use.

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.

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.