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Gaetano Donofrio - One of the best experts on this subject based on the ideXlab platform.
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Bovine Herpesvirus 4-based vector delivering the full length xCT DNA efficiently protects mice from mammary cancer metastases by targeting cancer stem cells.
Oncoimmunology, 2018Co-Authors: Gaetano Donofrio, Valeria Rolih, Giulia Tebaldi, Elisabetta Bolli, Francesca Macchi, Stefania Lanzardo, Roberto Ruiu, Andrea Ballatore, Laura Conti, Federica CavalloAbstract:Despite marked advancements in its treatment, breast cancer is still the second leading cause of cancer death in women, due to relapses and distal metastases. Breast cancer stem cells (CSCs), are a cellular reservoir for recurrence, metastatic evolution and disease progression, making the development of novel therapeutics that target CSCs, and thereby inhibit metastases, an urgent need. We have previously demonstrated that the cystine-glutamate antiporter xCT (SLC7A11), a protein that was shown to be overexpressed in mammary CSCs and that plays a key role in the maintenance of their redox balance, self-renewal and resistance to chemotherapy, is a potential target for mammary cancer immunotherapy. This paper reports on the development of an anti-xCT viral vaccine that is based on the Bovine Herpesvirus 4 (BoHV-4) vector, which we have previously showed to be a safe vaccine that can transduce cells in vivo and confer immunogenicity to tumor antigens. We show that the vaccination of BALB/c mice with BoHV-4 expressing xCT (BoHV-4-mxCT), impaired lung metastases induced by syngeneic mammary CSCs both in preventive and therapeutic settings. Vaccination induced T lymphocyte activation and the production of anti-xCT antibodies that can mediate antibody-dependent cell cytotoxicity (ADCC), and directly impair CSC phenotype, self-renewal and redox balance. Our findings pave the way for the potential future use of BoHV-4-based vector targeting xCT in metastatic breast cancer treatment.
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Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses.
Frontiers in immunology, 2018Co-Authors: Francesca Macchi, Sandro Cavirani, Valentina Franceschi, Giulia Tebaldi, José M. Rojas, Andrea Elizabeth Verna, Noemí Sevilla, Verónica Martín, Gaetano DonofrioAbstract:Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.
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Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells
Biology of reproduction, 2016Co-Authors: Giulia Tebaldi, Sarah Jacca, Alfonso Rosamilia, Simone Ottonello, Barbara Montanini, Emanuele Capra, Arianna Sala, Alessandra Stella, Bianca Castiglioni, Gaetano DonofrioAbstract:Viral infections can cause genital tract disorders (including abortion) in cows, and Bovine Herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of Bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a Bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation.
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Bovine Herpesvirus 4 based vector delivering a hybrid rat human her 2 oncoantigen efficiently protects mice from autochthonous her 2 mammary cancer
OncoImmunology, 2016Co-Authors: Sarah Jacca, Valentina Franceschi, Valeria Rolih, Elena Quaglino, Giulia Tebaldi, Elisabetta Bolli, Alfonso Rosamilia, Simone Ottonello, Federica Cavallo, Gaetano DonofrioAbstract:ABSTRACTThe epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of Bovine Herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes ...
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Bovine Herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer
Oncoimmunology, 2015Co-Authors: Sarah Jacca, Valentina Franceschi, Valeria Rolih, Elena Quaglino, Giulia Tebaldi, Elisabetta Bolli, Alfonso Rosamilia, Simone Ottonello, Federica Cavallo, Gaetano DonofrioAbstract:ABSTRACTThe epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of Bovine Herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes ...
Alain Vanderplasschen - One of the best experts on this subject based on the ideXlab platform.
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Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions
Journal of virology, 2012Co-Authors: Céline Lété, Alain Vanderplasschen, Leonor Palmeira, Baptiste Leroy, Jan Mast, Bénédicte Machiels, Ruddy Wattiez, Laurent GilletAbstract:GammaHerpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host’s antiviral response. In this study, we analyzed the protein composition of Bovine Herpesvirus 4 (BoHV-4), a close relative of the human Kaposi’s sarcoma-associated Herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaHerpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.
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Sequencing of Bovine Herpesvirus 4 V.test strain reveals important genome features
Virology journal, 2011Co-Authors: Leonor Palmeira, Céline Lété, Alain Vanderplasschen, Bénédicte Machiels, Laurent GilletAbstract:Bovine Herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaHerpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use. In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of Herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other Herpesviruses.
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Bovine Herpesvirus 4 immediate early 2 rta gene is an essential gene and is duplicated in Bovine Herpesvirus 4 isolate u
Veterinary Microbiology, 2011Co-Authors: Valentina Franceschi, Laurent Gillet, Alain Vanderplasschen, Sandro Cavirani, Vicky L. Van Santen, Antonio Capocefalo, Lara Ravanetti, Gaetano DonofrioAbstract:The ORF50/Rta gene has been shown to be an essential gene for many gammaHerpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U.
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The Bovine Herpesvirus 4 Bo10 Gene Encodes a Nonessential Viral Envelope Protein That Regulates Viral Tropism through both Positive and Negative Effects
Journal of virology, 2010Co-Authors: Bénédicte Machiels, Katalin De Fays, Alain Vanderplasschen, Benjamin G Dewals, Céline Lété, Jan Mast, Philip G. Stevenson, Laurent GilletAbstract:All gammaHerpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated Herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of Bovine Herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid Herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.
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Bovine Herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo.
The Journal of general virology, 2010Co-Authors: M Thirion, Benjamin G Dewals, G Donofrio, B Machiels, F Farnir, L Gillet, Alain VanderplasschenAbstract:ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine Herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue, with a size equivalent to only 22 % of that of the largest orthologues. The present study focused on determining whether BoHV-4 ORF73 is a bona fide gene and investigating whether it is essential for latency, as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early polycistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by the wild type and revertant recombinants. Together, these results demonstrate that, despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology.
Laurent Gillet - One of the best experts on this subject based on the ideXlab platform.
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Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions
Journal of virology, 2012Co-Authors: Céline Lété, Alain Vanderplasschen, Leonor Palmeira, Baptiste Leroy, Jan Mast, Bénédicte Machiels, Ruddy Wattiez, Laurent GilletAbstract:GammaHerpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host’s antiviral response. In this study, we analyzed the protein composition of Bovine Herpesvirus 4 (BoHV-4), a close relative of the human Kaposi’s sarcoma-associated Herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaHerpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.
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Sequencing of Bovine Herpesvirus 4 V.test strain reveals important genome features
Virology journal, 2011Co-Authors: Leonor Palmeira, Céline Lété, Alain Vanderplasschen, Bénédicte Machiels, Laurent GilletAbstract:Bovine Herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaHerpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use. In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of Herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other Herpesviruses.
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Bovine Herpesvirus 4 immediate early 2 rta gene is an essential gene and is duplicated in Bovine Herpesvirus 4 isolate u
Veterinary Microbiology, 2011Co-Authors: Valentina Franceschi, Laurent Gillet, Alain Vanderplasschen, Sandro Cavirani, Vicky L. Van Santen, Antonio Capocefalo, Lara Ravanetti, Gaetano DonofrioAbstract:The ORF50/Rta gene has been shown to be an essential gene for many gammaHerpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U.
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The Bovine Herpesvirus 4 Bo10 Gene Encodes a Nonessential Viral Envelope Protein That Regulates Viral Tropism through both Positive and Negative Effects
Journal of virology, 2010Co-Authors: Bénédicte Machiels, Katalin De Fays, Alain Vanderplasschen, Benjamin G Dewals, Céline Lété, Jan Mast, Philip G. Stevenson, Laurent GilletAbstract:All gammaHerpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated Herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of Bovine Herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid Herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.
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Establishment of a Bovine Herpesvirus 4 based vector expressing a secreted form of the Bovine Viral Diarrhoea Virus structural glycoprotein E2 for immunization purposes
BMC Biotechnology, 2007Co-Authors: Gaetano Donofrio, Laurent Gillet, Alain Vanderplasschen, Sandro Cavirani, Chiara Sartori, Simone Taddei, Lara Ravanetti, C. F. FlamminiAbstract:Background The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Results A recombinant Bovine Herpesvirus 4 (BoHV-4CMV-IgKE2-14ΔTK) expressing an enhanced secreted form of the Bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering ( recombi nation -mediated genetic engi neering ) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase – based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14ΔTK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14ΔTK, high levels of serum neutralized antibodies against BVDV were generated. Conclusion This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.
Sandro Cavirani - One of the best experts on this subject based on the ideXlab platform.
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Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses.
Frontiers in immunology, 2018Co-Authors: Francesca Macchi, Sandro Cavirani, Valentina Franceschi, Giulia Tebaldi, José M. Rojas, Andrea Elizabeth Verna, Noemí Sevilla, Verónica Martín, Gaetano DonofrioAbstract:Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.
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Bovine Herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg
BMC Veterinary Research, 2013Co-Authors: Valentina Franceschi, Sandro Cavirani, Antonio Capocefalo, Gaetano DonofrioAbstract:Background Bovine Herpesvirus 4 (BoHV-4) is a gammaHerpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection. Results The results show, however, that none of these soluble forms are able to block viral infectivity. To better understand the role of gB during BoHV-4 lytic replication, a recombinant BoHV-4 was generated by homologous recombination from a BoHV-4 cloned as a Bacterial artificial chromosome (BAC) (pBAC-BoHV-4-A), in which most of the BoHV-4 gB ORF was substituted by the insertion of a DNA stuffer selectable cassette. The resulting recombinant BoHV-4 genome (pBAC-BoHV-4-AΔgB-KanaGalK) was completely unable to reconstitute infectious replicating viral particles (Infectious Replicating Viral Particles, IRVPs) and to replicate when transfected in permissive cell lines in comparison to its revertant clone (pBAC-BoHV-4-ΔgB-Rev) or pBAC-BoHV-4-A parental clone. Conclusion This demonstrates that the BoHV-4 replicating cycle is dependent on gB. Moreover, when gB was deleted from a recombinant BoHV-4 genome delivering an heterologous glycoprotein, Vesicular Stomatitis Virus Glycoprotein (VSVg), VSVg was unable to complement gB. This study provides direct evidence that gB is necessary for BoHV-4 lytic replication.
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Bovine Herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg
BMC veterinary research, 2013Co-Authors: Valentina Franceschi, Sandro Cavirani, Antonio Capocefalo, Gaetano DonofrioAbstract:Background Bovine Herpesvirus 4 (BoHV-4) is a gammaHerpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection.
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Bovine Herpesvirus 4 immediate early 2 rta gene is an essential gene and is duplicated in Bovine Herpesvirus 4 isolate u
Veterinary Microbiology, 2011Co-Authors: Valentina Franceschi, Laurent Gillet, Alain Vanderplasschen, Sandro Cavirani, Vicky L. Van Santen, Antonio Capocefalo, Lara Ravanetti, Gaetano DonofrioAbstract:The ORF50/Rta gene has been shown to be an essential gene for many gammaHerpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U.
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Swine adipose stromal cells loaded with recombinant Bovine Herpesvirus 4 virions expressing a foreign antigen induce potent humoral immune responses in pigs.
Vaccine, 2010Co-Authors: Gaetano Donofrio, Sandro Cavirani, Antonio Capocefalo, Valentina Franceschi, S. Taddei, Simone Ottonello, Nicola Martinelli, Maura FerrariAbstract:Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by Bovine Herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from Bovine viral diarrhea virus (BVDV) and Bovine Herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.
C. F. Flammini - One of the best experts on this subject based on the ideXlab platform.
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Cellular targeting of engineered heterologous antigens is a determinant factor for Bovine Herpesvirus 4-based vaccine vector development.
Clinical and vaccine immunology : CVI, 2009Co-Authors: Gaetano Donofrio, Sandro Cavirani, Antonio Capocefalo, Valentina Franceschi, S. Taddei, Chiara Sartori, Sabrina Bonomini, Clotilde Silvia Cabassi, C. F. FlamminiAbstract:In a previous study, an apathogenic strain of Bovine Herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against Bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.
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assessment of Bovine Herpesvirus 4 based vector in chicken
Journal of Virological Methods, 2008Co-Authors: Gaetano Donofrio, C. F. Flammini, Sandro Cavirani, Lara Ravanetti, G Manarolla, G Sironi, C S Cabassi, T RampinAbstract:The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.
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Establishment of a Bovine Herpesvirus 4 based vector expressing a secreted form of the Bovine Viral Diarrhoea Virus structural glycoprotein E2 for immunization purposes
BMC Biotechnology, 2007Co-Authors: Gaetano Donofrio, Laurent Gillet, Alain Vanderplasschen, Sandro Cavirani, Chiara Sartori, Simone Taddei, Lara Ravanetti, C. F. FlamminiAbstract:Background The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Results A recombinant Bovine Herpesvirus 4 (BoHV-4CMV-IgKE2-14ΔTK) expressing an enhanced secreted form of the Bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering ( recombi nation -mediated genetic engi neering ) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase – based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14ΔTK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14ΔTK, high levels of serum neutralized antibodies against BVDV were generated. Conclusion This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.
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Bovine Herpesvirus 4 is tropic for Bovine endometrial cells and modulates endocrine function
Reproduction (Cambridge England), 2007Co-Authors: Gaetano Donofrio, C. F. Flammini, Sandro Cavirani, Chiara Sartori, Shan Herath, Iain Martin SheldonAbstract:Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the gamma-Herpesvirus Bovine Herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.
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Generation of a transposon insertion mutant library for Bovine Herpesvirus 4 cloned as a bacterial artificial chromosome by in vitro MuA based DNA transposition system
Journal of virological methods, 2006Co-Authors: Gaetano Donofrio, Alain Vanderplasschen, C. F. Flammini, Sandro Cavirani, Chiara Sartori, Eugenio Martignani, Laurent GilletAbstract:Bovine Herpesvirus 4 (BoHV-4) is a gammaHerpesvirus with no clear disease association. Although the BoHV-4 genome has been sequenced, the function of the majority of putative genes is elusive. Several features make BoHV-4 attractive as a backbone for use as a viral expression vector and/or as a model to study gamma Herpesvirus biology and determining which genes are essential for its replication is a very important task. Starting from BoHV-4 genome cloned as infectious bacterial artificial chromosome (BAC-BoHV-4) in Escherichia coli. A random insertion mutant library for BoHV4 was generated by the use of MuA transposase-catalyzed in vitro transposition reaction. Viral mutant transfection and direct sequencing allow the rapid determination of which BoHV-4 genes are essential for viral growth in a permissive eukaryotic cell line. BoHV-4 functional analysis information is fundamental when the BoHV-4 genome is modified for vector purposes.