Antitumor activity and immunogenicity of recombinant vaccinia virus expressing HPV 16 E7 protein SigE7LAMP is enhanced by high-level coexpression of IGFBP-3.

We constructed recombinant vaccinia viruses (VACVs) coexpressing the insulin-like growth factor-binding protein-3 (IGFBP-3) gene and the fusion gene encoding the SigE7Lamp antigen. The expression of the IGFBP-3 transgene was regulated either by the early H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that IGFBP-3 expression regulated by the H5 promoter yielded higher amount of IGFBP-3 protein when compared with the E/L promoter. The immunization with P13-SigE7Lamp-H5-IGFBP-3 virus was more effective in inhibiting the growth of TC-1 tumors in mice and elicited higher T-cell response against VACV-encoded antigen than the P13-SigE7Lamp-TK(-) control virus. We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in more profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7Lamp-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7Lamp-TK(-). Intracellular mature virions (IMVs) of the IGFBP-3-expressing virus P13-SigE7Lamp-H5-IGFBP-3 have two structural differences: they incorporate the IGFBP-3 protein and they have elevated phosphatidylserine (PS) exposure on outer membrane that could result in increased uptake of IMVs by macropinocytosis. The IMV PS content was measured by flow cytometry using microbeads covered with immobilized purified VACV virions.

Expression of soluble TGF-ß receptor II by recombinant Vaccinia virus enhances E7 specific immunotherapy of HPV16 tumors.

Therapeutic immunization with double recombinants of vaccinia virus (VACV) co-expressing sTßRII increased rejection of established TC-1 tumors in C57BL/6 mice in comparison with single recombinant expressing SigE7LAMP. Recombinant VACV derived from vaccination strain Praha expressed either the sTßRII (ectodomain) or chimeric protein fused to immunoglobulin Fc fragment (sTßRII-Fc-Jun) under control of two different promotors together with the immunogenic tumor associated antigen HPV16 E7 oncoprotein in a form of SigE7LAMP fusion molecule. The ability of soluble receptors to bind TGF-ß in vitro was proved. Immunization of mice with double recombinant viruses and virus expressing SigE7LAMP only led to eliciting similar response of E7 specific CD8+ T cells as detected by IFN-γ ELISPOT.

The expression of the soluble isoform of hFlt3 ligand by recombinant vaccinia virus enhances immunogenicity of the vector.

Recombinant vaccinia viruses (rVACV) expressing various tumor-associated antigens have been shown to elicit anti-tumor effect in numerous experimental models and clinical trials. We tested the hypotheses that rVACV expressing biologically active fms-like tyrosine kinase 3 ligand (Flt3L) would show higher immunogenicity than control viruses expressing only model antigen and that coexpression of Flt3L would influence anti-tumor activity of rVACV in the preventive and therapeutic arrangements of the in vivo experiment. To answer these questions, we took advantage of the well-described model of transplanted tumor cells expressing HPV16 E6 and E7 oncoproteins. To determine the effects of hFlt3L on the induction of anti-tumor immunity, we generated live vaccinia viruses that express human Flt3L regulated by the early H5 or strong synthetic E/L promoter together with fusion protein SigE7LAMP, which is a highly immunogenic form of HPV E7 oncoprotein. We tested Flt3L production in vitro and in vivo. Despite higher expression of Flt3L from the synthetic E/L promoter in vitro, the P13-E/L-FL-SigE7LAMP induced lower levels of Flt3L in the serum of mice than P13-H5-FL-SigE7LAMP. The Flt3L expression under the strong early VACV H5 promoter is able to inhibit expansion of CD11b+Gr-1+ myeloid suppressor cells (MSC) and increase the amount of CD11b+ CD11c+ dendritic cells in the spleen of mice immunized with vaccinia virus. Determination of viral DNA isolated from the ovaries of infected animals did not reveal differences in replication between rVACVs in this organ. Coexpression of Flt3L by replication-competent virus P13-FL-SigE7LAMP induced enhancement of the cellular immune response against HPV16 E7 and VACV E3 proteins as well as increased anti-tumor efficacy in both the protective and therapeutic immunization schemes. On the other hand, the short-time Flt3L coexpression by MVA-H5-FL-SigE7LAMP was not sufficient to enhance anti-tumor effect of immunization.

Combination of intratumoral injections of vaccinia virus MVA expressing GM-CSF and immunization with DNA vaccine prolongs the survival of mice bearing HPV16 induced tumors with downregulated expression of MHC class I molecules.

Downregulation of MHC class I molecules is believed to be often the cause of tumor immune escape and at the same time it is the major obstacle to T-cell based immunotherapy of tumors. In our experimental model, the C57BL/6 mice bearing tumors induced by TC-1/A9 cells characterized by expression of HPV16 oncogenes and downregulation of H-2b molecules were immunized with highly immunogenic E7GGG.GUS DNA vaccine expressing the fused gene of modified HPV16 E7 (E7GGG) with E.coli beta-glucuronidase (GUS). The DNA vaccine was administered by gene gun on days 7 and 14 after s.c. injection of tumor cells. The tumors in situ were injected with recombinant vaccinia virus MVA expressing the gene for murine granulocyte-macrophage colony-stimulating factor (MVA-GM-CSF). Two doses of the DNA vaccine combined with at least two consecutive local treatments with MVA-GM-CSF were able to inhibit significantly the growth of tumors. We have shown by ELISPOT-IFNgamma that in situ expression of the GM-CSF gene did not enhance the E7 specific systemic Tcell response. We found that local injections of MVA-GM-CSF induced an increase of intratumoral CD3+ T cell counts and that the DNA vaccination resulted in up-regulation of MHC type I molecules on tumor cells in vivo. We suppose that i.t. delivery of MVA-GM-CSF changed the local tumor microenvironment and rendered tumors more attractive and better accessible to effector T cells.

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.).

[Vaccinia virus as a vector for transduction of dendritic cells]. / Virus vakcinie jako vektor pro transdukci dendritických bunek.

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.

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.

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.

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.

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.

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.

Transient transformation of mammalian cells by MN protein, a tumor-associated cell adhesion molecule with carbonic anhydrase activity.

The MN protein is associated with certain human carcinomas, but absent in most normal tissues. It is a transmembrane protein; its extracellular part contains a domain homologous with carbonic anhydrases (CAs) and a proteoglycan-like region. In the present study, we observed that cells (human CGL1 and mouse NIH3T3 cells) transfected with MN cDNA showed morphologic transformation, but reverted to normal phenotype after 4-5 weeks. This reversion was not due to the loss, silencing, or mutations of MN insert. We also found that MN protein exerted CA enzymatic activity, but this was not relevant for morphologic transformation of cells. MN is an adhesion protein, involved in cell-to-cell contacts, this probably could explain its role in tumorigenesis.

Hepatitis B virus core-preS2 particles expressed by recombinant vaccinia virus.

Vaccinia virus (VV) recombinants expressing hepatitis B virus (HBV) surface (HBsAg) or core (HBcAg) antigens (Kunke et al., Virology 195, 132 - 139 (1993)] have been shown to raise specific antibodies in mice, nevertheless the levels of antibodies reactive with the preS2 and S antigens were low. In an attempt to enhance the immunogenicity of HBsAg-preS2, a fused C-preS2 gene was constructed. The fusion protein was expressed in E. coli and displayed both HBcAg and preS2 antigen as demonstrated by enzyme-linked immunosorbent assay (ELISA). The same gene was then expressed using recombinant VV and chimerical particles whose size and density were similar to those of native HBV core particles produced in CV-1 cells infected with recombinant VV. Unlike HBcAg, preS2 antigen could not be detected on these particles by ELISA but was revealed by immunoblot analysis only. The immunogenicity of the recombinant VV was evaluated in mice. Antibodies to HBcAg and VV antigen but not to preS2 antigen were found in sera of animals inoculated with 10(7) PFU of the recombinant VV. Presumably, HBcAg-preS2 particles produced in E. coli and in eukaryotic cells have a different conformation, and the presence of preS2 antigen on the surface of chimerical particle might be necessary for a pronounced antibody response.

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.

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.

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.

[Construction of recombinant vaccinia viruses]. / Konstrukce rekombinant viru vakcínie.

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.

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.

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.

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.