Biomedical subjects

L Kutinová

Publications and source records attributed to L Kutinová.

At least 19 recordsLinked to original sources

PubMed2003

Immunization with Varicella-zoster virus glycoprotein E expressing vectors: Comparison of antibody response to DNA vaccine and recombinant vaccinia virus.

Immunization with DNA vaccines expressing Varicella-zoster virus (VZV) glycoprotein E (gE) induced formation of specific antibodies in mice. The antibody response correlated with the level of in vitro gE expression if the plasmid was inoculated intradermally (i.d.) with a gene gun but not if intramuscular (i.m.) injection was used. The i.d. vaccination produced a higher antibody level than the i.m. one even though a 100-fold amount of DNA was administered. A plasmid expressing a truncated form of gE was less immunogenic. The magnitude of antibody response induced by immunization with recombinant vaccinia viruses (rVVs) was equivalent to the gene gun vaccination. Administration of DNA by i.m. route or Vaccinia virus (VV) gE by i.d. mute resulted in predominance of IgG2a in the response while the gene gun plasmid inoculation usually elicited similar levels of IgG1 and IgG2a. The antibody response elicited by DNA vaccine was boosted by a secondary immunization with rVV. The boosting effect was highest if the virus was administered intraperitoneal (i.p.).

Animals↗

PubMed2002

[Vaccinia virus as a vector for transduction of dendritic cells].

Dendritic cells (DC) are very heterogenous population of professional antigen-presenting cells. Precursor cells migrate from bone marrow to peripheral tissues, where immature DC ingest pathogenic microorganisms and then migrate to secondary lymphoid organs. DC differentiate into mature cells that are capable to prime naive T lymphocytes. DC can be used for immunotherapy of cancer and infectious diseases. Transduction of DC by recombinant viral vectors expressing tumor associated antigens (TAA) can result in efficient antigen presentation to T lymphocytes. DC transduced with recombinant vaccinia virus expressing E7 oncoprotein of human papilloma virus 16 are able to protect mice against the growth of syngeneic papillomavirus transformed tumor cells TC1. Antitumor effect was observed also with nonreplicating viruses.

Animals↗

PubMed2001

Immune response to vaccinia virus recombinants expressing glycoproteins gE, gB, gH, and gL of Varicella-zoster virus.

Immunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs.

Animals↗

PubMed2001

Early gene expression of vaccinia virus strains replicating (Praha) and non-replicating (modified vaccinia virus strain Ankara, MVA) in mammalian cells.

Modified vaccinia virus Ankara strain (MVA) is a safe highly attenuated non-pathogenic virus suitable as a vector for developing various vaccines. Study of expression of a reporter beta-galactosidase gene under the control of an early vaccinia virus (VV) promoter in MVA and non-modified vaccinia virus Praha strain showed that early transcription in MVA is elevated in comparison with non modified VV. This property was demonstrated in various cell cultures including CV1 cells, human lung diploid cells, chicken primary fibroblasts but not in bone marrow-derived mouse dendritic cells. There the relationship between the elevated early transcription and the permisivity of cells for MVA was not observed.

Animals↗

PubMed2000

Characterization of interaction of gH and gL glycoproteins of varicella-zoster virus: their processing and trafficking.

Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system. Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide. Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed. The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa). Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa). Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together. The secreted form of gL was 18 kDa while that of gH was 114 kDa. The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion. The 19 kDa form of gL was never secreted. These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Biological Transport↗

PubMed1999

Secondary vaccination with vaccinia virus recombinants: role of residual virulence of recombinants and immunogenicity of extrinsic antigens.

ICR mice were immunized intraperitoneally with two doses (10(6) PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Aging↗

PubMed1998

Effect of 3-beta-hydroxysteroid dehydrogenase gene deletion on virulence and immunogenicity of different vaccinia viruses and their recombinants.

3-beta-Hydroxysteroid dehydrogenase (3-beta-HSD) activity coded for by the A44L gene of vaccinia virus (VV) was demonstrated in CV-1 cultures infected by all five VV strains tested, viz. WR, Praha virus, DRYVAX Wyeth-derived virus (DD), LIVP and MVA. Deletion of the A44L gene in two Praha virus-derived clones (the moderately virulent P13 and the highly attenuated P20), the WR and DD viruses resulted in absence of 3-beta-HSD activity from infected cultures. The virulence for mice of P13 was not affected, and that of WR was only slightly decreased, by the A44L gene deletion. Recombinant VVs expressing either varicella-zoster virus glycoprotein E (VZV-gE) or hepatitis B virus preS2-S protein (HBV-preS2-S) and their respective A44L deleted mutants were used in immunogenicity tests in mice. In terms of antibody response to VV and the recombinant proteins, the deletion resulted in a lowering the immunogenicity in the moderately virulent clone P13 virus and its progenies. In the highly attenuated P20 and DD viruses and their progenies no effects were apparent.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Influence of the parental virus strain on the virulence and immunogenicity of recombinant vaccinia viruses expressing HBV preS2-S protein or VZV glycoprotein I.

Five triple-plaque purified vaccinia virus (VV) lines generated from smallpox Sevac VARIE vaccine (strain Praha) and three VV virus lines similarly derived from Wyeth DRYVAX vaccine were used for preparation of recombinants expressing the hepatitis B virus preS2-S gene. The same five Praha-derived virus lines were used to construct recombinants expressing the varicella-zoster virus (VZV) glycoprotein I (gpI) gene. Recombinants and their parental viruses were tested for the residual neurovirulence in mice. The virus lines and the recombinants derived therefrom differed markedly in this respect. Immunization of mice resulted in high levels of anti-HBsAg antibodies only in the case of recombinants derived from the relatively virulent viruses. In contrast, the levels of VZVgpI antibodies in mice were similar with all VV-VZV recombinants irrespective of the virulence of the parental virus line.

Animals↗

PubMed1996

Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene.

Recombinant vaccinia viruses (VV) expressing the varicella-zoster virus (VZV) glycoprotein H (gH) or glycoprotein L (gL) were constructed. The 94 kDa gH intermediate glycoprotein was synthesized in cell cultures infected with the VV-gH recombinant, but only co-infection with both recombinants resulted in the synthesis of the fully processed 118 kDa gH molecule. The VV-expressed gH and gL formed a complex that displayed the conformational neutralization epitope detectable by means of human VZV gH-specific monoclonal antibody V3. Formation of this epitope was inhibited by tunicamycin but not by monensin. Simultaneous intraperitoneal inoculation of mice with high doses of both VV-gH and VV-gL viruses resulted in the development of VZV-neutralizing, complement-independent antibodies; these antibodies were not detected in mice infected solely with either the VV-gH or the VV-gL recombinant.

Animals↗

PubMed1995

Search for optimal parent for recombinant vaccinia virus vaccines. Study of three vaccinia virus vaccinal strains and several virus lines derived from them.

Three vaccinia virus strains (Praha, DD--a DRYVAX Wyeth vaccine-derived virus-and LIVP) were examined for growth in various cell cultures and for virulence and immunogenicity in mice. The viruses did not differ by their growth rates in monkey kidney cells (CV-1), human diploid cells (LEP), rat TK cells (RAT 2) or primary dog kidney cells. The immunogenicity of Praha and DD viruses was similar, the virus LIVP was somewhat more immunogenic. In terms of virulence in 3-day-old mice, the DD virus was the most attenuated. Single-plaque progenies were derived from the original smallpox vaccines VARIE Sevac (strain Praha) and DRYVAX Wyeth and tested for the above markers and DNA restriction patterns. The results obtained demonstrated biological and molecular heterogeneity of the original virus populations. Close linkage was observed between immunogenic activity and virulence in 3-day but not in 3-week mice. The results indicate that smallpox vaccine preparations may serve as an abundant source of virus mutants.

3T3 Cells↗

PubMed1994

[Construction of recombinant vaccinia viruses].

Expression system based on vaccinia virus (VV) is used both for recombinant protein production in vitro and as an alive vaccine. The article summarizes various strategies for recombinant VV construction, and describes preparation of recombinants expressing various forms of S and C genes of hepatitis B virus (HBV). It is shown, that the CV-1 cells infected with these recombinants synthesized the MS (middle) and the LS (large) polypeptides of the surface antigen (HBsAg) and the nucleocapsid antigen (HBcAg) polypeptide and the polypeptide HBeAg. Posttranslation modification of all expressed proteins was the same as at HBV infection.

Hepatitis B↗

PubMed1994

A carboxy-terminal portion of the preS1 domain of hepatitis B virus (HBV) occasioned retention in endoplasmic reticulum of HBV envelope proteins expressed by recombinant vaccinia viruses.

The large envelope glycoprotein (L protein) of Hepatitis B virus (HBV) contains the preS1 domain, which is responsible for retention of the protein in the endoplasmic reticulum. To identify sequences of the preS1 domain involved in this phenomenon we constructed vaccinia virus-HBV recombinants containing the gene for L protein in which the preS1 coding sequence had been partially deleted. The retention of L protein in the endoplasmic reticulum was found to be mediated by a sequence contained within a region of 35 amino acids of the preS1 C-terminus, and not exclusively by amino acid sequences of the N-terminus of the preS1 domain as proposed by Kuroki et al. (Mol. Cell. Biol. 9, 4459-4466, 1989). Our finding could be explained by a specifically VV promoter sequence leading to exclusive synthesis of L or deleted (delta)L proteins, respectively. The ability of the coexpressed HBV S protein to facilitate export of the delta L proteins was demonstrated by coinfection experiments.

Base Sequence↗

PubMed1994

Evaluation of various virulence tests with low virulence vaccinia virus in mice.

Two vaccinia virus (VV) clones, denoted P20 and P21 were derived from the parental VV strain Praha. The residual virulence of these two viruses in mice was compared. Intracerebral inoculation of 21-day-old mice did not reveal any marked difference between the two viruses. Tests using intracerebral inoculation of 10-day-old mice showed that their LD50 differed by 2 log10 pfu, P20 being the more attenuated. The difference between the two viruses increased further when intranasal and intracerebral inoculations of 3-day-old mice were employed. Their LD50 differed by 4 and 5 log10 pfu, respectively. Thus, these tests proved to be the most sensitive for determining residual virulence of VV in mice and thus the most suitable for differentiating among the low virulence VVs. The antibody tests carried out in the surviving mice suggested that the more attenuated virus was less immunogenic than the more virulent virus.

Animals↗

PubMed1994

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells.

Fifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine-derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

Animals↗

PubMed1993

Vaccinia virus recombinants co-expressing hepatitis B virus surface and core antigens.

Using the Praha strain of vaccinia virus (VV) two double recombinant VVs expressing the surface and capsid HBV proteins (HBsAg and HBcAg) under the control of the P7.5 promoter were constructed. In the first construct the gene coding for HBsAg was inserted into the HindIII J fragment (TK gene) and the gene coding for HBcAg was inserted into the HindIII M fragment (host range, K1L gene) of the VV genome. To test whether the expression of the foreign genes was influenced by the insertion site, in the second construct their locations were inversely changed. When compared with single VV-HBV recombinants expressing either HBsAg or HBcAg, the double recombinants expressed in vitro approximately the same amounts of the respective antigens. The particles formed by either HBsAg or HBcAg expressed by recombinant viruses, were isolated and examined by electron microscopy. Particles composed of both HBsAg and HBcAg were not detected in cultures infected with one of the double recombinants. The residual virulence in 3-week-old mice of the single recombinants was not markedly altered by the insertion of the second gene. The immunogenicity in mice of both the single and double recombinants was comparable and was not influenced by the location of the HBV genes in VV genome, as revealed by antibodies developed against the respective antigens.

Animals↗

PubMed1992

[Problems associated with the development of vaccines based on recombinant vaccinia virus].

A collection of single and double vaccinia virus recombinants was prepared. The recombinants contained either genes for different forms of surface protein or core protein of HBV or the gene for glycoprotein I of varicella-zoster virus. Cells infected with the recombinants produced the respective foreign antigens. Specific antibodies against the heterologous antigens were induced in mice immunized with the recombinants. The insertion of the second foreign gene into the genome of single recombinants did not influence either the extent of production of the first protein in vitro or its immunogenicity for mice.

Animals↗

PubMed1991

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus. Finding of retention signal in the C-terminal portion of the preS1 domain of subtype adyw.

Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P7.5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.

Animals↗

PubMed1991

Immunogenicity in mice of varicella-zoster virus glycoprotein I expressed by a vaccinia virus-varicella-zoster virus recombinant.

Several vaccinia virus (VV)-varicella-zoster virus (VZV) recombinants expressing glycoprotein I (gpI) of VZV were isolated from the Prague strain of VV. One of these, v46, was inoculated intraperitoneally into mice. Groups of mice were bled 4 and 8 weeks later and their sera were examined for anti-VZV and anti-VV antibodies by ELISA. At 4 weeks, all mice inoculated with the three largest virus doses (10(7), 10(6) and 10(5) p.f.u.), and at 8 weeks all mice inoculated with the four highest virus doses (10(7), 10(6), 10(5), and 10(4) p.f.u.), had developed both anti-VV and anti-VZV antibodies. Antibodies were also detected in a high proportion of mice infected with lower doses of virus and in some instances VZV antibodies were present in the absence of VV antibodies. None of the animals inoculated in parallel with either a thymidine kinase-negative mutant of the original VV or diluent alone developed antibody reactive with VZV. The specificity of the reaction was assessed further by Western blotting using anti-gpI monoclonal antibodies as a positive control. Sera from animals immunized with v46 possessed antibody capable of neutralizing extracellular VZV in the presence of complement.

Animals↗