BoHV-4-based vector delivering Ebola virus surface glycoprotein

Background: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106TK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106TK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106TK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106TK viremia and secondary localization was detected in any of the immunized animals. Conclusions: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications

Bovine Endometrial Stromal Cells Support Tumor Necrosis Factor Alpha-Induced Bovine Herpesvirus Type 4 Enhanced Replication

Bovine uterine infections are the most important cause of economic losses in the cattle industry. Although the etiology of uterine diseases is mainly ascribed to bacterial infection, they can also be associated with viral infection, such as bovine herpesvirus 4 (BoHV-4), which is often a secondary agent following bacteria. Besides microbial infection, many inflammatory molecules belonging to the innate immune response orchestrate the outcome of the infection. In the present study, the interaction between BoHV-4-infected bovine endometrial stromal cells and tumor necrosis factor alpha (TNF-alpha) was investigated. Bovine herpesvirus 4 possesses a special tropism toward endometrial stromal cells. For this reason, a simian virus 40 (SV40) immortalized endometrial stromal cell line (SV40BESC) was established; it was proven that it was stable, it expressed toll-like receptors (TLRs; from 1 to 10) and TNFalpha receptors I and II, and it was responsive to exogenous TNF-alpha. Further, an increase of BoHV-4 replication and cytopathic effect was observed in BoHV-4-infected and TNFalpha- treated SV40BESCs. This increase of viral replication was associated with BoHV-4 immediate early 2 (IE2) gene promoter trans-activation through the interaction of the nuclear factor KB (NFKB) with the putative NFKB-responsive elements found within BoHV-4 IE2 gene promoter, and this interaction was abolished when NFKB-responsive elements were deleted. These data shed light on two important and rather controversial issues: the role of TNF-alpha receptor, which is weakly expressed in the stromal layer of the bovine uterus, as well as the possible interactions between proinflammatory molecules, viral replication, and chronic uterine disease

Bovine Endometrial Stromal Cells Support Tumor Necrosis Factor Alpha-Induced Bovine Herpesvirus Type 4 Enhanced Replication1

Bovine uterine infections are the most important cause of economic losses in the cattle industry. Although the etiology of uterine diseases is mainly ascribed to bacterial infection, they can also be associated with viral infection, such as bovine herpesvirus 4 (BoHV-4), which is often a secondary agent following bacteria. Besides microbial infection, many inflammatory molecules belonging to the innate immune response orchestrate the outcome of the infection. In the present study, the interaction between BoHV-4-infected bovine endometrial stromal cells and tumor necrosis factor alpha (TNF-alpha) was investigated. Bovine herpesvirus 4 possesses a special tropism toward endometrial stromal cells. For this reason, a simian virus 40 (SV40) immortalized endometrial stromal cell line (SV40BESC) was established; it was proven that it was stable, it expressed toll-like receptors (TLRs; from 1 to 10) and TNFalpha receptors I and II, and it was responsive to exogenous TNF-alpha. Further, an increase of BoHV-4 replication and cytopathic effect was observed in BoHV-4-infected and TNFalpha- treated SV40BESCs. This increase of viral replication was associated with BoHV-4 immediate early 2 (IE2) gene promoter trans-activation through the interaction of the nuclear factor KB (NFKB) with the putative NFKB-responsive elements found within BoHV-4 IE2 gene promoter, and this interaction was abolished when NFKB-responsive elements were deleted. These data shed light on two important and rather controversial issues: the role of TNF-alpha receptor, which is weakly expressed in the stromal layer of the bovine uterus, as well as the possible interactions between proinflammatory molecules, viral replication, and chronic uterine disease

Efficient heterologous antigen gene delivery and expression by a replication-attenuated BoHV-4-based vaccine vector

Bovine Herpesvirus 4 (BoHV-4) is a gammaherpesvirus belonging to the Rhadinovirus genus and due to its biological characteristics has been proposed as a vaccine vector for veterinary vaccines. Because viral vector-associated risk is a major concern for viral vector applications, attenuation is a desirable feature. Therefore, efforts are directed toward the development of highly attenuated viral vectors. BoHV-4 naturally exhibits limited pathogenicity and a further attenuation, in terms of replication, was obtained by disrupting the late gene encoding the 1.7-kb polyadenylated RNA (L1.7). An L1.7 deleted mutant BoHV-4 (BoHV-4-A-KanaGalKΔL1.7), as well as its revertant (BoHV-4-A-Rev), was generated by homologous recombination from the genome of a BoHV-4 isolate (BoHV-4-A) cloned as a bacterial artificial chromosome (BAC). BoHV-4-A-KanaGalKΔL1.7 showed attenuation in terms of competence to reconstitute infectious virus, viral replication, and plaque size when compared to BoHV-4-A, BoHV-4-A-Rev, and BoHV-4-A-KanaGalKΔTK, a recombinant control virus where the KanaGalK selectable marker was inserted into the thymidine kinase open reading frame. The capability of BoHV-4-A-KanaGalKΔL1.7 to deliver and express a heterologous antigen was investigated by replacing the KanaGalK cassette with a vesicular stomatitis virus glycoprotein (VSVg) expression cassette to generate BoHV-4-A-EF1αVSVgΔL1.7. BoHV-4-A-EF1αVSVgΔL1.7 infected cells robustly expressed VSVg, thus confirming that the replication deficiency resulting from L1.7 disruption did not prevent heterologous gene delivery and expression. Although further work is needed to identify the specific function of the BoHV-4 L1.7 gene, the L1.7 gene may represent an ideal targeting locus for the integration of a heterologous antigen expression cassette, resulting in attenuation of the viral vector

BoHV-4 immediate early 1 gene is a dispensable gene and its product is not a bone marrow stromal cell antigen 2 counteracting factor

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus whose genome was cloned as Bacterial Artificial Chromosome (BAC) and exploited as a gene delivery vector for vaccine purposes. Although BoHV-4 genome has been completely sequenced and its open reading frames (ORFs) structurally defined in silico, most of them are not functionally characterized. In BoHV-4 genome two major immediate early genes (IE) are present, IE1 and IE2. IE2 is an essential gene because its removal from the viral genome renders the virus unable to replicate, whereas for IE1 no many functional information are available. RESULTS: In this work, IE1 contribution in initiating and maintaining BoHV-4 lytic replication was assessed generating a recombinant BoHV-4 genome lacking of IE1 gene, BoHV-4ΔIE1. In contrast to BoHV-4IE2 deleted mutant, BoHV-4ΔIE1 infectious replicating viral particles (IRVPs) could be reconstituted following viral DNA electroporation in permissive cells. However the titer of BoHV-4ΔIE1 IRVPs produced into the cell supernatant and BoHV-4ΔIE1 plaques size were reduced respect to BoHV-4 undeleted control. Further the impaired BoHV-4ΔIE1 IRVPs produced into the cell supernatant could be rescued by expressing IE1 gene product in trans, confirming the implication of IE1 in BoHV-4 lytic replication. Next, the possible role of BoHV-4IE1 as bone marrow stromal cell antigen 2 (BST-2) counteracting factor, as hypothesized by IE1 amino-terminal gene product homology with Kaposi Sarcoma Associated Herpesvirus (KSHV) K5, was excluded too. CONCLUSIONS: Although the real function of BoHV-4IE1 is still elusive, a new BoHV-4 genome gene locus as a target site for the insertion of foreign DNA and resulting in the attenuation of the virus has been revealed. These data can be considered of relevance to improve BoHV-4 gene delivery properties. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-015-0540-4) contains supplementary material, which is available to authorized users

In vivo image analysis of BoHV-4-based vector in mice.

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice

International audiencePolymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer

The 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 at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs. © 2016 The Author(s). Published with license by Taylor & Francis Group, LL