Re-determination of the primary shelf-life of food products: what are the guarantees for the consumer?

Except in rare cases, the determination of the shelf-life of food products is left up to the food business operator. The extension of this period, which for years has been the subject of debate among the various actors in the food chain, has become a topic of fundamental importance also following the recent economic/financial, environmental, and health crises, which have had an inevitable impact on consumption and food waste. While there is no requirement to indicate durability for some categories of food products, for example, those not directly intended for consumers, this debate has raised questions and perplexities about the potential reevaluation of the origin conditions established by the manufacturer, particularly when it comes to maintaining the guarantees for the consumer in terms of health and hygiene. In addition, the increasing consumer demand for accurate information has prompted the European authorities to request a public consultation on the actual understanding and perception of the mandatory terms on labels such as use by or date of minimum durability of a food, provided for by Article 9 of Regulation (EU) No. 1169/2011, often not correctly understood, which can assume great significance in the application of rules to reduce food waste. In this regard, it is useful to remember that the recent measures adopted by the European Union legislator, together with the case law of recent years, have led the judges of merit to comply with the principles and requirements of food safety laid down since 2002 in Regulation (EC) No. 178, thus paying greater attention to the analysis, assessment, and management of the risk of the entire production chain. The purpose of this work is to provide technical-legal elements to encourage a possible extension of the shelf-life of food products while ensuring the safety of consumers

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

Generation and characterization of BoHV-4 based vector delivering Ebola surface glycoprotein

Il virus dell’Ebola (EBOV) è un patogeno di categoria A, membro della famiglia Filoviridae, responsabile di una malattia emorragica molto grave, spesso ad esito letale che colpisce l’uomo e la scimmia. Negli anni intercorsi fra il 1976 ed oggi, si sono verificate numerose epidemie della malattia in Africa occidentale. Il gran numero di casi e l’elevato tasso di mortalità, unitamente alla mancanza di trattamenti approvati, hanno creato la necessità di una nuova terapia e, ancor più, di un vaccino protettivo. Inoltre, uno dei limiti principali di lavorare con un virus come l’EBOV è la necessità di operare in strutture di contenimento molto costose. Lo scopo del presente lavoro è stato quello di esplorare una nuova strategia basata sull’utilizzo di Bovine Herpesvirus 4 (BoHV-4) come vettore virale a fini vaccinali. BoHV-4 è un Gammaherpesvirus caratterizzato da una bassa patogenicità e un ampio spettro d’ospite, sia in vitro che in vivo; inoltre, esso non sembra avere nessun potenziale oncogenico. Il genoma di BoHV-4 è stato clonato come Cromosoma Artificiale Batterico (BAC) e mostra, quindi, la potenzialità di trasportare geni codificanti antigeni di patogeni differenti; lo stesso ad oggi è stato già testato su diversi modelli animali, mostrando una buona attivazione della risposta immunitaria. Nel nostro studio è stato generato un gene sintetico, syEBOVgD106, codificante per la glicoproteina immunodominante (GP) di EBOV, ed è stato inserito mediante ricombinazione omologa all’interno del BAC di BoHV-4. Il BAC è stato in seguito trasfettato in cellule permissive per la ricostituzione del virus. Nelle cellule infettate con BoHV-4-syEBOVgD106ΔTK l’EBOV GP è stata abbondantemente espressa senza evidenti cambiamenti nella cinetica di replicazione virale. Negli esperimenti realizzati in vivo, mediante inoculo del virus in un modello animale come la capra, è stata confermata la stimolazione di una buona risposta anticorpale attraverso un test ELISA. Le capre immunizzate con BoHV-4-syEBOVgD106ΔTK hanno prodotto alti titoli di anticorpi anti-EBOV GP, conferendo una risposta immunitaria di tipo umorale rilevabile persino oltre sei mesi dall’inoculo del virus. BoHV-4-syEBOVgD106ΔTK non ha dato viremia e neppure una localizzazione secondaria del virus negli animali immunizzati. La strategia vaccinale, basata sull’utilizzo di geni sintetici e di una piattaforma vaccinale derivata dal virus BoHV-4, rappresenta un sistema alternativo di espressione di un antigene per la sperimentazione di vaccini verso patogeni di categoria A. Inoltre, l’elevato titolo anticorpale, rilevato fino a sei mesi dopo la prima vaccinazione nelle capre, così come la sicurezza e l’efficienza del vettore stesso, rendono BoHV-4 un eccellente candidato nell’immunizzazione sperimentale delle capre, che possono essere utilizzate come animali siero-produttori.Ebola virus (EBOV) is a Category A pathogen, a member of the Filoviridae family responsible for a very serious haemorrhagic disease, often with a lethal outcome affecting human and monkey. In the years between 1976 and today, there have been several epidemics of disease in West Africa. The large number of cases and the high mortality rate along with the lack of approved treatments have created the need of new therapies and even more of protective vaccines. Moreover, one of the main limitations of working with virus such as EBOV is the need to operate in very costly containment structures. The purpose of this paper was to explore a new therapeutic strategy based on Bovine Herpesvirus 4 (BoHV-4) as a viral vector for vaccine applications. BoHV-4 is a Gammaherpesvirus characterized by low pathogenicity and a broad spectrum of hosts, both in vitro and in vivo. Moreover, it does not seem to have any oncogenic potential. The BoHV-4 genome has been cloned as an Bacterial Artificial Chromosome (BAC) with the potential to carry antigen-encoding genes of different pathogens and has already been tested on several animal models, showing a good activation of the immune response. In our study a synthetic gene syEBOVgD106, coding for EBOV immunogenic glycoprotein (GP), was developed and inserted by homologous recombination within BoHV-4 BAC. The BoHV-4 BAC was subsequently transfected into permissive cells for the reconstitution of the virus. BoHV-4-syEBOVgD106ΔTK EBOV infected cells abundantly expressed the GP without detrimental effects on viral replication. Experiments were carried out in vivo, by inoculating the virus into a large animal model, stimulation of a good antibody response was confirmed by means of an ELISA assay. Goats immunized with BoHV-4-syEBOVgD106ΔTK produced high titres of anti-EBOV GP antibodies, giving a humoral immune response detectable up to six months from the inoculum of the virus. No BoHV-4-syEBOVgD106ΔTK viremia or even secondary location of the virus was detected in immunized animals. This vaccinal strategy exploiting synthetic genes using a BoHV-4-based vaccine platform is an alternative antigen delivery system to test vaccines against Category A pathogens. Moreover, high antibody titre, detected up to six months after the first goat vaccination, as well as safety and efficacy, make BoHV-4 an excellent candidate for experimental goat immunization, which can be used as serum-producing animals

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

BoHV-4-based vector delivering Ebola virus surface glycoprotein

Abstract 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-syEBOVgD106ΔTK, was generated. Results EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK 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-syEBOVgD106ΔTK 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 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

Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells

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

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

Alignment between KSHV K5 and BoHV-4IE1 protein sequence. The sequence of KSHV K5 protein was aligned with BoHV-4 Immediate early 1 gene product to highlight the percentage of sequence identities and positive matches through the alignment software BLAST-2 ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=blasttab ). (TIFF 5192 kb