Galina V. Kochneva

Individual Escherichia coli Cells Studied from Light Scattering with the Scanning Flow Cytometer

BACKGROUND Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles.

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.

Effects of deletions of kelch -like genes on cowpox virus biological properties

Summary.Cowpox virus (CPXV) strain GRI-90 contains six genes encoding kelch-like proteins. All six proteins contain both, the N-terminal BTB domain and the C-terminal kelch domain. We constructed mutant variants of a CPXV strain with targeted deletions of one to four genes of the kelch family, namely D11L, C18L, G3L, and A57R. As kelch genes are located in terminal variable regions of the CPXV genome, we studied the relationship of these genes with integral biological characteristics such as virulence, host range, reproduction in vitro and in ovo (in chicken embryos). It was demonstrated that the following effects occurred in a gene dose dependent manner with an increase of the number of genes deleted: (1) range of sensitive cells altered – deletion mutants lacking three genes displayed a considerably decreased ability to reproduce in MDCK cells; mutants lacking four genes lost this ability completely; (2) analysis of pocks formed by mutants with deletion of three and four kelch-like genes on chorioallantoic membranes of chicken embryos demonstrated that pock size and virus yield were significantly decreased; (3) light microscopic analysis of the pocks revealed impaired proliferation and reduced vascularisation in the pock region. More alterations were detected by electron microscopic analysis: the reproduction of mutants results in a reduction of the number of mature virions formed, and in many cells this process was arrested at the stage of assembly of immature virions; and (4) the evaluation of LD50 and body weight loss in BALB/c mice infected intranasally with CPXVs revealed a reduction of the virulence of the deletion mutants, which became statistically significant when four kelch-like genes were excised.

Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence.

Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype). Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation. The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice. In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation. Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates. Thus, the mouse acts as a live filter much more efficiently than any other selective systems. Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible. Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants. Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.

Features of the Antitumor Effect of Vaccinia Virus Lister Strain

Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, “natural” oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells.

Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population.

Chronic hepatitis C is a severe liver disease caused by positive-strand RNA virus. Previously, we reported an association between seven single nucleotide polymorphisms (SNPs) in four innate immunity genes (OAS2, OAS3, CD209, and TLR3) and human predisposition to tick-borne encephalitis, caused by a virus from the same Flaviviridae family, in a Russian population. Currently, genotype and allele frequencies for these SNPs were analyzed in 75 chronic hepatitis C patients and compared with the population control (269 Novosibirsk citizens). Data obtained suggest that the OAS2 rs1293762 and CD209 rs2287886 SNPs are associated with predisposition to chronic hepatitis C in Russian population.

Engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy

Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase −3 and −7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings.

Complete Genome Sequence of the Oncolytic Sendai virus Strain Moscow

ABSTRACT We report here the complete genome sequence of Sendai virus Moscow strain. Anecdotal evidence for the efficacy of oncolytic virotherapy exists for this strain. The RNA genome of the Moscow strain is 15,384 nucleotides in length and differs from the nearest strain, BB1, by 18 nucleotides and 11 amino acids.

Complete Genome Sequence of Vaccinia Virus Strain L-IVP.

ABSTRACT Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%.

Antitumor Effect of Vaccinia Virus Double Recombinant Strains Expressing Genes of Cytokine GM-CSF and Oncotoxic Peptide Lactaptin

In this study, the double recombinant vaccinia viruses were generated those express exogenous proteins: human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the antitumor protein lactaptin in secreted and nonsecreted forms. We observed that recombinant VV-GMCSF-Lact with nonsecreted lactaptin exerted stronger cytotoxic activity than others in MDA-MB-231, BT-549 and BT-20 breast cancer cells with calculated CD50 of 0.005; 0.004 и 0.00083 PFU/cell correspondently. Strain VV-GMCSF-Lact also exhibited highest lytic activity in lung cancer cells H1299 and epidermoid carcinoma cells A-431. Normal MCF10A cells and diploid embryonic lung human cells LECH-240 were resistant to all recombinant vaccinia viruses. Strain VV-GMCSF-Lact showed the highest index of tumor selectivity in pairs normal/cancer cells: MCF10A/MDA-MB-231 (>2000) and LECH-240/H1299 (190). By flow cytometry, we demonstrated that all recombinants induced apoptosis in treated cancer cells but the rate of annexin V-positive cells was higher after treatment with VV-GMCSF-Lact than others. Thus nonsecreted lactaptin expression increased the toxicity of recombinant virus to cancer cells in the best way. It is likely that lactaptin expression inside the treated cells (without secretion outside) intensifies apoptosis and as a consequence promotes the progression of apoptotic cells to secondary necrotic cells. These results demonstrate that recombinant VV-GMCSF-Lact has good oncolytic potential and stimulate further investigation of its anticancer activity in human tumor models in vivo and to use it in the development of anticancer therapeutic agents.