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1.
Epidemiol Mikrobiol Imunol ; 68(3): 144-148, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31914780

RESUMEN

Adoptive immunotherapy using antiviral T cells (AVT) obtained from healthy donors is one of the advanced approaches considered as a breakthrough in the treatment of refractory and severe viral infections that often accompany primary immunodeficiencies or allogeneic hematopoietic stem cell transplantations. The review describes nearly 30 years of the development of AVT to human cytomegalovirus, Epstein-Barr virus, human adenovirus, and human polyomavirus BK. The review introduces the basic methodological approaches to their production and summarizes the results from clinical studies that tested the safety and efficiency of the procedures used. Recent studies indicate that the treatment of viral infections by frozen AVT stored in banks could become a commonly available therapeutic modality.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva , Linfocitos T , Receptores de Trasplantes , Virosis , Antivirales/inmunología , Humanos , Linfocitos T/inmunología , Virosis/terapia
2.
Transpl Infect Dis ; 18(3): 381-9, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27061389

RESUMEN

BACKGROUND: The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. METHODS: T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. RESULTS: We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. CONCLUSION: The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.


Asunto(s)
Antígenos Virales/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Proteínas Virales/inmunología , Antígenos Virales/genética , Antivirales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Citomegalovirus/genética , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , Ensayo de Immunospot Ligado a Enzimas , Femenino , Humanos , Huésped Inmunocomprometido , Interferón gamma/sangre , Masculino , Persona de Mediana Edad , Fosfoproteínas/inmunología , Proteínas Virales/genética
3.
Transpl Infect Dis ; 18(2): 297-301, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26858009

RESUMEN

BACKGROUND: Human cytomegalovirus (HCMV) establishes lifelong latent infection that can result in severe life-threatening disease in immunosuppressed patients after hematopoietic stem cell transplantation (HSCT). An HCMV-seropositive transplant recipient who receives a graft from a seronegative donor (R+/D-) is at high risk of recurrent HCMV reactivation. METHODS: To assess the incidence of R+/D- combination, we retrospectively evaluated HCMV-seronegative donors for 746 allogeneic HSCT treatments carried out at our center during 1995-2014. In our cohort, 20% HCMV-seronegative HSCT recipients, 21% HCMV-seronegative related graft donors, and 52% HCMV-seronegative unrelated graft donors were included. RESULTS: Analyses of the HCMV serostatus of hematopoietic stem cell donors during 2 consecutive calendar periods (1995-2005 and 2006-2014) showed a significant increase in the proportion of seronegative donors (odds ratio [OR] = 1.947). In addition, the number of HSCT treatments using an unrelated donor increased (OR = 2.376). Finally, the use of grafts from countries with a very low HCMV prevalence increased. CONCLUSION: This increase in HCMV seronegativity in unrelated donors and the increased proportion of unrelated donors were responsible for the increased occurrence of the high-risk combination R+/D- (OR = 1.680). If the reduction in the rate of HCMV-seropositive graft donors continues, an increased frequency of HCMV reactivation events in our transplant recipients can be expected, because of the increasing occurrence of the high-risk R+/D- combination.


Asunto(s)
Infecciones por Citomegalovirus/epidemiología , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Activación Viral/inmunología , República Checa/epidemiología , Humanos , Huésped Inmunocomprometido , Estudios Retrospectivos , Donantes de Tejidos
4.
Cancer Gene Ther ; 21(3): 115-25, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24556712

RESUMEN

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.


Asunto(s)
Antígenos Virales/inmunología , Papillomavirus Humano 16/inmunología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas E7 de Papillomavirus/inmunología , Virus Vaccinia/inmunología , Vacunas Virales/inmunología , Animales , Formación de Anticuerpos , Femenino , Papillomavirus Humano 16/genética , Inmunización/métodos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas E7 de Papillomavirus/genética , Regiones Promotoras Genéticas , Linfocitos T/inmunología , Vacunación/métodos , Virus Vaccinia/genética , Vacunas Virales/farmacología , Replicación Viral/inmunología
5.
Neoplasma ; 58(3): 181-8, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21391733

RESUMEN

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.


Asunto(s)
Papillomavirus Humano 16/inmunología , Neoplasias Experimentales/terapia , Proteínas E7 de Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Animales , Femenino , Inmunización , Ratones , Ratones Endogámicos C57BL , Receptor Tipo II de Factor de Crecimiento Transformador beta , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia/genética
6.
Oncol Rep ; 21(5): 1335-43, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19360312

RESUMEN

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.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Proteínas de la Membrana/biosíntesis , Virus Vaccinia/inmunología , Análisis de Varianza , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Transfección , Virus Vaccinia/genética
7.
Neoplasma ; 54(4): 326-33, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17822323

RESUMEN

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.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Antígenos H-2/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/mortalidad , Vacunas de ADN/administración & dosificación , Virus Vaccinia/genética , Animales , Vacunas contra el Cáncer/administración & dosificación , Regulación hacia Abajo , Escherichia coli/enzimología , Femenino , Terapia Genética , Glucuronidasa , Papillomavirus Humano 16/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasia Residual/etiología , Neoplasia Residual/patología , Neoplasia Residual/terapia , Proteínas E7 de Papillomavirus , Tasa de Supervivencia , Vacunación
8.
Acta Virol ; 47(1): 1-10, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12828337

RESUMEN

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.).


Asunto(s)
Anticuerpos Antivirales/sangre , Vectores Genéticos , Herpesvirus Humano 3/inmunología , Vacunas contra Herpesvirus/genética , Vacunas contra Herpesvirus/inmunología , Virus Vaccinia/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Células Cultivadas , Femenino , Regulación Viral de la Expresión Génica , Herpesvirus Humano 3/genética , Vacunas contra Herpesvirus/administración & dosificación , Inmunización , Ratones , Ratones Endogámicos ICR , Modelos Genéticos , Pruebas de Neutralización , Proteínas Recombinantes/inmunología , Transfección , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo
9.
Cas Lek Cesk ; 141 Suppl: 23-5, 2002 Sep 22.
Artículo en Checo | MEDLINE | ID: mdl-12428418

RESUMEN

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.


Asunto(s)
Antígenos de Neoplasias/genética , Células Dendríticas/inmunología , Vectores Genéticos , Transducción Genética , Virus Vaccinia , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos de Neoplasias/metabolismo , Humanos
10.
Arch Virol ; 146(2): 239-49, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11315635

RESUMEN

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.


Asunto(s)
Eliminación de Gen , Receptores de Interferón/fisiología , Virus Vaccinia/fisiología , Virus Vaccinia/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops , Cartilla de ADN , Humanos , Interferón gamma/fisiología , Ratones , Reacción en Cadena de la Polimerasa , Conejos , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Vaccinia/inmunología , Virulencia , Receptor de Interferón gamma
11.
Virology ; 280(2): 211-20, 2001 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11162835

RESUMEN

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.


Asunto(s)
Antígenos Virales/inmunología , Herpesvirus Humano 3/inmunología , Glicoproteínas de Membrana/inmunología , Virus Vaccinia/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Línea Celular , Chlorocebus aethiops , Proteínas del Sistema Complemento/inmunología , Femenino , Expresión Génica , Vectores Genéticos/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Cobayas , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiología , Humanos , Glicoproteínas de Membrana/genética , Ratones , Recombinación Genética , Piel/inmunología , Pruebas Cutáneas , Proteínas del Envoltorio Viral/genética , Ensayo de Placa Viral , Proteínas Virales/genética
12.
Int J Mol Med ; 7(1): 97-100, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11115616

RESUMEN

Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus , Infecciones Tumorales por Virus/prevención & control , Secuencia de Aminoácidos , Animales , Humanos , Inmunoterapia Adoptiva , Ratones , Ratones Endogámicos C57BL , Infecciones por Papillomavirus/virología , Péptidos/síntesis química , Péptidos/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Células Tumorales Cultivadas , Infecciones Tumorales por Virus/virología , Vacunas Sintéticas/inmunología
13.
Acta Virol ; 45(4): 243-7, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11885931

RESUMEN

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.


Asunto(s)
Virus Vaccinia/genética , Animales , Northern Blotting , Línea Celular , Células Cultivadas , Embrión de Pollo , Chlorocebus aethiops , Expresión Génica , Vectores Genéticos , Humanos , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/análisis , ARN Viral/análisis , Recombinación Genética , Transcripción Genética , Virus Vaccinia/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genética
15.
J Gen Virol ; 81(Pt 6): 1545-52, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10811938

RESUMEN

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.


Asunto(s)
Herpesvirus Humano 3/metabolismo , Glicoproteínas de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Virales/metabolismo , Transporte Biológico , Medios de Cultivo , Glicosilación , Herpesvirus Humano 3/genética , Humanos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/genética
16.
Vaccine ; 17(9-10): 1186-92, 1999 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-10195631

RESUMEN

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.


Asunto(s)
Anticuerpos Antivirales/biosíntesis , Antígenos Virales/inmunología , Inmunización Secundaria , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Vacunas Virales/inmunología , Envejecimiento/inmunología , Animales , Antígenos Virales/genética , Embrión de Pollo , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Antígenos de Superficie de la Hepatitis B/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Precursores de Proteínas/inmunología , Virus Vaccinia/inmunología , Proteínas del Envoltorio Viral/inmunología
17.
Arch Virol ; 143(7): 1311-20, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-9722876

RESUMEN

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.


Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Eliminación de Gen , Genes Virales , Virus Vaccinia/enzimología , Virus Vaccinia/genética , Animales , Anticuerpos Antivirales/biosíntesis , Línea Celular , Cartilla de ADN/genética , ADN Viral/genética , ADN Viral/aislamiento & purificación , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Ratas , Recombinación Genética , Especificidad de la Especie , Virus Vaccinia/patogenicidad , Virulencia/genética
18.
Acta Virol ; 40(5-6): 273-9, 1996.
Artículo en Inglés | MEDLINE | ID: mdl-9171455

RESUMEN

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.


Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Precursores de Proteínas/inmunología , Vacunas Sintéticas/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Vectores Genéticos , Antígenos de la Hepatitis B/biosíntesis , Antígenos de la Hepatitis B/genética , Antígenos de la Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Immunoblotting , Inmunogenética , Ratones , Ratones Endogámicos ICR , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virus Vaccinia
19.
Vaccine ; 14(11): 1045-52, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8879101

RESUMEN

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.


Asunto(s)
Anticuerpos Antivirales/biosíntesis , Antígenos de Superficie de la Hepatitis B/biosíntesis , Precursores de Proteínas/biosíntesis , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/inmunología , Animales , Línea Celular , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Antígenos de Superficie de la Hepatitis B/inmunología , Herpesvirus Humano 3/metabolismo , Ratones , Ratones Endogámicos ICR , Precursores de Proteínas/inmunología , Especificidad de la Especie , Virus Vaccinia/genética , Virulencia
20.
J Gen Virol ; 77 ( Pt 2 ): 211-5, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8627224

RESUMEN

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.


Asunto(s)
Anticuerpos Antivirales/biosíntesis , Herpesvirus Humano 3/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunología , Animales , Embrión de Pollo , Humanos , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Virus Vaccinia/genética
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