Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination.

AIM: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. METHODS: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. RESULTS: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. CONCLUSION: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.

[Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.

[Specific clinical, epidemiological patterns and laboratory diagnostics of enterovirus infection in the Republic of Belarus].

The clinical and epidemiological patterns as well as the results of the laboratory verification of the outbreak of enterovirus infection (EVI) in Minsk during the period of summer-autumn, 2000, are presented. During this outbreak a variety of clinical forms were observed, the serous meningitis being prevalent (57.5%). Practically simultaneous occurrence of infection on the territory of all administrative districts of the city, the predominant involvement of children aged up to 14 years into the outbreak, a high proportion of simultaneous casualities in the multiple foci. A number of circulating enteroviruses (EV)--ECHO 30, ECHO 6 of three serotypes and Coxsackie B5--were simultaneously isolated from clinical material. EV of the same serotypes were isolated from tap drinking water, and neutralizing antibodies to these serotypes were often detected in the patients blood sera. Infectious EV were also present in samples of bottled water and in water reservoirs used for bathing. The routes of EV transmission and the improvement of EVI control are discussed.

[Molecular biological monitoring of the polioviruses circulation in Belarus]. / Molekuliarno-biologicheskii monitoring tsirkuliatsii poliovirusov v Belarusi.

A total of 135 polioviruses (PV), including 25 wild and 110 vaccine-related, isolated in Belarus in 1957-1999 were studied by the analysis of the polymorphism of the restriction fragments lengths of two distal regions of the genome: the region (480 oligonucleotide pairs) coding the N-terminal fragment of capsid protein VP1 (RLFP-1) and the region (291 oligonucleotide pairs) coding the N-terminal fragment of nonstructural protein of 3D-polymerase (RLFP-3D1). The genetic analysis of the viruses made it possible to determine 3 epidemiologically different periods of PV circulation: (1) the prevaccination period (1957-1959) when wild PV of all 3 serotypes circulated on the territory of Belarus; (2) the early period of the use of Oral Poliomielytis Vaccine (1960-1966), characterized by simultaneous circulation of wild and vaccine PV, as well as vaccine/wild recombinant PV; (3) the period of the elimination of wild PV of indigenous origin and the circulation of vaccine-related viruses (1967-1999). The characteristic feature of wild PV was their pronounced genetic variability. 8 genetic variants of PV1, including 4 genetic groups, 2 genetic variants of PV2 and 1 genetic variant of PV3 were detected; 2 vaccine/wild recombinant PV were detected in 1960 and 1966. More than 40% of the vaccine-related PV under study had altered genetic characteristics (mutations and/or recombinations. Reverse variability, linked with the loss of a number of signs of attenuation, was shown to be characteristic of vaccine PV1. Recombinants occurred most frequently among PV3 (44.9%) and PV2 (40.0%), their recombinations being formed mainly with PV1. Recombinants PV2/PV1 and PV3/PV1 were found to have high frequency of reversion in the "PV1" fragment of the genome; this frequency exceeded that in PV1 with the homotypical genome (66.7 and 44.4% in contrast to 12.5%).

[Mutation and recombination variability of vaccine polioviruses isolated in Belarus (1960-1999)]. / Mutatsionnaia i rekombinatsionnaia izmenchivost' vaktsinnykh poliovirusov, izolirovannykh v Belarusi (1960-1999 gg.).

One hundred and eight vaccine-derived strains of types 1, 2, and 3 poliovirus (25 PV1, 34 PV2, and 48 PV3) isolated in Belarus in 1960-1999 were analyzed by double restriction fragment length polymorphism assay (RFLP-1, -3D1). Forty-four (40.7%) of strains were genetically modified. Eight (7.4%) PV were modified by mutation, 16 (14.8%) by recombination, and 20 (18.5%) by both mutation and recombination. The genomes of 16 PV were analyzed by multiple RFLP technique covering VP1-, VP1/VP2A-, P2-, 3AC-, and 3D1-coding regions. The majority of recombinants were "simple" (with one crossing over site). One strain was "double" recombinant (two crossings over sites) and one more "multiple" recombinant (three crossing over sites). Partial nucleotide sequencing of some recombinant strains showed that the degree of these strains' divergence was less than 1% in comparison with the original vaccine viruses.

[Isolation and characteristics of low molecular weight recombinant peptides, representing antigenic domains in the NP protein of lymphocytic choriomeningitis virus]. / Poluchenie i kharakteristika nizkomolekuliarnykh rekombinantnykh peptidov, predstavliaiushchikh antigennye domeny belka NP virusa limfotsitarnogo khoriomeningita.

High- and low-molecular recombinant peptides of LCM virus nucleoprotein, representing individual immunodominant antigenic sites, were obtained in pJC40 expressing vector and studied in solid-phase enzyme immunoassay. 2-7% proteins resultant from total cellular synthesis are recombinant peptides. Comparative analysis of antigenic properties of recombinant peptides and native viral protein showed that recombinant peptides are virtually not inferior to native viral protein in antigenic properties.

[False-positive reactions in laboratory diagnosis of Lassa, Marburg, and Ebola viral hemorrhagic fevers and AIDS]. / Lozhnopolozhitel'nye reaktsii pri laboratornoi diagnostike virusnykh gemorragicheskikh likhoradok Lassa, Marburg, Ebola i SPIDa.

Sera of normal subjects and AIDS patients living in Minsk and Odessa were tested for antibodies to hazardous viral infections Lassa, Marburg, and Ebola. Four to 16% of examinees were seropositive to Ebola virus, 0.8 to 2.3% to Lassa, and up to 0.8% to Marburg virus. Common B-epitopes were found in viruses belonging to different families: Lassa, Ebola, and HIV. Antibodies specific to these viruses antigens were found in the reference sera to influenza A and B, respiratory syncytial virus, and adenovirus. Sera of convalescents after malaria and of AIDS patients contained antibodies to Lassa virus.

[The isolation and characteristics of reassortants between the Lassa and Mopeia arenaviruses]. / Poluchenie i kharakteristika reassortantov mezhdu arenavirusami Lassa i Mopeiia.

Reassortants with a mixed phenotype were produced by combined inoculation of Vero cells with Lassa and Mopeya viruses. These reassortants produced small plaques (Mopeya virus phenotype) and were not pathogenic for newborn mice (Lassa virus phenotype). The genotype of the reassortants was studied by dot hybridization experiments on filters using cDNA-probes differentiating genome segments of these viruses. The reassortants were shown to have Mopeya virus L-RNA and Lassa virus S-RNA.

[Virus-specific proteins and RNA of the virus of hemorrhagic fever with renal syndrome]. / Virusspetsificheskie belki i RNK virusa gemorragicheskoi lokhoradki s pochechnym sindromom.

Virus-specific proteins G1, G2, and N with molecular weights of 70, 55-57, and 50 kilodaltons, respectively, were detected by radioimmunodiffusion tests in VERO E-6 cells infected with strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the European USSR from a patient with HFRS, a fatal human case of HFRS (the strains K-27 and P-360) and from a bank vole (strain CG-1820). The sera from human convalescents after HFRS in the European USSR and rat sera prepared with the CG-1820 strain precipitated proteins possessing similar electrophoretic characteristics from a lysate of cells infected with the CG-1820, K-27 and P-360 strains. The sera from human HFRS convalescents in the Far East did not precipitate protein Gl. The viral RNA derived by immunosorption method from intracellular nucleocapsids of CG-1820 strain and strain 4590 (isolated from Ap. Peninsulae in the Far East) contained 3 classes of molecules: L, M, and S. L- and M-RNA of these strains had the same molecular weight (1.62 and 1.38 megadaltons). The molecular weight of S-RNA of the strain 4590 was 0.76 megadalton and that of the CG-1820 strain 0.83 megadalton. It is assumed that there are species differences among the viruses, causative agents of HFRS, circulating in the European and Far East regions of the USSR.

[Template activity of poly(A+) and poly(A-) messenger RNA of pathogenic arenaviruses]. / Matrichnaia aktivnost' poli(A+) i poli(A-) informatsionnykh RNK patogennykh arenavirusov.

The total RNA from cells infected with Machupo and Lassa viruses as well as poly(A+) and poly(A-) fractions of the RNA were translated in the cell-free protein synthesizing system from rabbit reticulocytes. The translated products were treated with specific antibodies and analyzed in polyacrylamide gel electrophoresis. Only poly(A-) fraction of RNA coded for the synthesis of NP protein in vitro. The mRNAs for NP protein of Machupo and Lassa viruses are supposed to contain no poly(A) sequences at 3'end, or if they really do, the size of the sequences is not adequate for binding with oligo(dT)-cellulose.

[Identification of the mRNA of the nucleocapsid proteins of pathogenic arenaviruses]. / Identifikatsiia mRNK belkov nukleokapsida patogennykh arenavirusov.

Total RNA from cells infected with Machupo and Lassa viruses as well as individual sedimentation classes of these RNAs were translated in the cell-free protein-synthesizing system from rabbit reticulocytes. The translation products were precipitated either with anti-Machupo immune gamma-globulin or monoclonal antibodies to nucleocapsid protein (NP) of Lassa virus. Both total RNA and RNA fraction with the sedimentation coefficient 15-16S promoted the synthesis of protein which comigrated in gel with NP protein of purified virions. It is concluded that monocistron mRNA for NP protein of arenaviruses has the sedimentation coefficient 15-16 S.

[Sedimentation characteristics of virion RNA of Machupo virus reproducing in the presence of actinomycin D]. / Sedimentatsionnaia kharakteristika virionnykh RNK virusa Machupo, reprodutsiruiushchegosia v prisutstvii aktinomitsina D.

Actinomycin D treatment (0.005-05 g/ml) of Vero and BHK-21 cells infected with Machupo virus suppressed the synthesis of ribosomal RNAs but did not affect the production of infectious Machupo virus. Virion RNAs contained 3 high molecular weight RNA species: 28-31 S, 22-24 S and 18 S. In the presence of actinomycin D [3H]-uridine incorporated only in 30-31 S and 22-24 S RNA species. The data are supported by previous results which show that Machupo virus genome contains two RNA species: "large" (30-31 S) and "small" (22-24 S).