M. Ortega-Villaizan

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

In addition to its antiviral and immunomodulatory properties, the zebrafish β-defensin 2 (zfBD2) is a potent viral DNA vaccine molecular adjuvant

It is well known that β-defensins are key components of the host innate immune response against pathogens and potentially provide a link between innate and adaptive immunity. In zebrafish (Danio rerio), a vertebrate model species in numerous biomedical fields, three β-defensin isoforms were recently identified. To our knowledge, however, studies describing antimicrobial or immunomodulatory properties of any of the zebrafish β-defensins isoforms are absent today. Since it is indubitable that deepening the study of zebrafish β-defensins would be of interest in this work we investigated whether or not the zebrafish β-defensin 2 (zfBD2) has the antiviral properties described for their vertebrate counterparts. Our in vitro and in vivo studies showed that zfBD2 has antiviral activity, immunomodulatory properties and, most importantly, is a potent viral DNA vaccine molecular adjuvant. In addition, a potential relationship between zfBD2 activity and the NF-κB signaling pathway is suggested. Altogether these results show that the zebrafish could be a suitable in vivo animal model to study the roles played by β-defensin 2 in viral diseases, vaccinology and even in clinical dermatology. To note that psoriasis can be induced in zebrafish and the over-expression of β-defensin 2 is implicated in the inflammatory response associated with this human skin disorder.

VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants

We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpGVHSV), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpGVHSV. In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31+p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1μg of the construct pMCV1.4-p31+p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31+p33 construct was assayed for molecular adjuvantcitys for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-GSVCV). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-Gsvcv without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system.

Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration

Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5µg) at 2days before infection (1.1×107) held at 26°C died even though mortality was delayed from 8days (1µg) and 4days (5µg). Similarly, CpG ODN 1668 administered (5µg) at 2days before infection (1.2×106) held at 23/20°C had 100% mortality; the mortality was delayed from 9days (23°C) and 11days (20°C). Moreover, when CpG ODN 1668 administered (1/5/10µg) at 2/4/7days before infection or virus concentration was decreased to 1.1×104 and held at 20°C had mortality rates of 20/60/30% (2days), 30/40/60% (4days) and 60/60/20% (7days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1×107) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20°C and infection dose of 1.1×104).

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.

Increasing Versatility of the DNA Vaccines through Modification of the Subcellular Location of Plasmid-Encoded Antigen Expression in the In Vivo Transfected Cells

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1-507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1-462) or a secreted soluble (gpGLmPle20-462) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpGLmPle20-462 (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates

DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.