Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of ß-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus).

Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV(860)) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G(860)). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV(860) lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization.

Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major lineal determinants implicated in triggering host cell antiviral responses mediated by type I interferon.

Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

Antimicrobial peptides as model molecules for the development of novel antiviral agents in aquaculture.

Antimicrobial peptides (AMPs) are one of the components of the non-specific immune system that operate first lines of protection in many animal species including fish. They exert broad-spectrum antimicrobial activity, apart from many other potential roles in innate immunity, and represent a promising class of antiviral agents. Recent advances in understanding the mechanisms of their antiviral action(s) indicate that they have a dual role in antiviral defence, acting not only directly on the virion but also on the host cell. Despite the acute problems of viral diseases and restrictions in using chemicals in aquaculture, few but successful attempts to assess the antiviral activities of fish AMPs have been reported. This review focuses on the antiviral activities and mechanisms of action of some AMPs, and their potential relevance in the aquaculture industry, one of the most important sources of fishery products in the near future. It is a matter of notable concern to understand whether the AMPs can be used as model molecules for designing antiviral drugs that might help to solve the problems with viruses in the fish farming industry worldwide. In addition, because fish rely more heavily on their innate immune defences than mammals, they might constitute a potential rich source of antiviral compounds for fighting against mammalian viral infections.

Transfection improvements of fish cell lines by using deacylated polyethylenimine of selected molecular weights.

A new tool for DNA transfer to fish cell lines such as epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG2), has been optimized by testing commercially available polyethylenimine (PEI) polymers as transfectant reagents. Deacylated 25 kDa PEI polymers were selected amongst the most active and then low toxicity deacylated PEIs fractions around 15 kDa were obtained by gel filtration chromatography to increase 3-4-fold their initial in vitro transfection efficiency. The EPC and plasmids coding for reporter genes were first used to optimize variable values for best expression by transfection with deacylated low toxicity PEI while both EPC/RTG2 and a plasmid coding for the glycoprotein G gene of the fish pathogen, viral haemorrhagic septicemia virus (VHSV) were then used to demonstrate some of their practical applications. Due to its relatively low price, defined chemical composition and availability, low toxicity deacylated PEI might be used for numerous applications for all those studying fish cell immunology in vitro as well as in vivo.

Expression and antiviral activity of a beta-defensin-like peptide identified in the rainbow trout (Oncorhynchus mykiss) EST sequences.

The in silico identification of a beta-defensin-like peptide sequence (omBD-1) in the rainbow trout (Oncorhynchuss mykiss) database of salmonid EST is reported here. We have studied the transcript expression of this beta-defensin-like sequence in different organs and expressed the recombinant peptide in a fish cell line. Finally, we have demonstrated the in vitro antiviral activity of the recombinant trout beta-defensin-like peptide against viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most devastating viruses for worldwide aquaculture. Thus, the resistance to VHSV infection of EPC cells transfected with pMCV 1.4-omBD-1 has been shown. Since EPC cells transfected with omBD-1 produced acid and heat stable antiviral activity and up regulation of Mx, a type I IFN-mediated mechanism of antiviral action is suggested. To our knowledge, this is the first report showing biological activity of a beta-defensin-like peptide from any fish.

Dual antiviral activity of human alpha-defensin-1 against viral haemorrhagic septicaemia rhabdovirus (VHSV): inactivation of virus particles and induction of a type I interferon-related response.

It is well known that human alpha-defensin-1, also designated as human neutrophil peptide 1 (HNP1), is a potent inhibitor towards several enveloped virus infecting mammals. In this report, we analyzed the mechanism of the antiviral action of this antimicrobial peptide (AMP) on viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Against VHSV, synthetic HNP1 possesses two antiviral activities. The inactivation of VHSV particles probably through interfering with VHSV-G protein-dependent fusion and the inhibition of VHSV replication in target cells by up-regulating genes related to the type I interferon (IFN) response, such as Mx. Neither induction of IFN-stimulated genes (ISGs) by HNP1 nor their antiviral activity against fish rhabovirus has been previously reported. Therefore, we can conclude that besides to acting as direct effector, HNP1 acts across species and can elicit one of the strongest antiviral responses mediated by innate immune system. Since the application of vaccine-based immunization strategies is very limited, the used of chemicals is restricted because of their potential harmful impact on the environment and no antimicrobial peptides from fish that exhibit both antiviral and immunoenhancing capabilities have been described so far, HNP1 could be a model molecule for the development of antiviral agents for fish. In addition, these results further confirm that molecules that mediate the innate resistance of animals to virus may prove useful as templates for new antivirals in both human and animal health.

Low 25-hydroxyvitamin D concentrations in obese women: their clinical significance and relationship with anthropometric and body composition variables.

UNLABELLED: Obesity is associated with low concentrations of 25-hydroxyvitamin D [25(OH) D]. However, conflicting results have been found regarding the relationship of 25(OH) D with anthropometric and adiposity parameters. The aim of our study was to analyze the association between 25(OH) D and body fat (BF) in a homogeneous cohort of non-obese, obese, and morbidly obese Caucasian women. The study was performed in L'Hospitalet de Llobregat, a city adjacent to Barcelona with a latitude of 41 degrees, 22 minutes, and 5 seconds north. MATERIALS AND METHODS: Plasma concentrations of 25(OH) D were determined and body composition was evaluated by bioelectrical impedance in a group of 43 women with morbid obesity, 28 non-morbidly obese, and 50 non-obese women matched for age. RESULTS: Morbidly obese women showed lower 25(OH) D concentrations compared to non-morbidly and non-obese women (37.9+/-16 vs 40.2+/-13 vs 56.7+/-21 nmol/l, p=0.001). Fifty-one percent of morbidly obese women had vitamin D deficiency [25(OH) D<38 nmol/l] compared to 22% of non-obese patients, (p=0.004). In the bivariate correlation analysis 25(OH) D was inversely associated with weight (r=-0.41, p=0.001), body mass index (BMI) (r=-0.432, p=0.001), waist to hip ratio (WHR)(r=-0.40, p=0.001), BF (r=-0.53, p=0001), fat mass (r=-0.44, p=0.0001), fat-free mass (r=-0.35, p=0.001). In the multivariate general linear model analysis, 25(OH) D was associated with season of examination (p=0.001) and was negatively associated with BF (beta=-0.75, p=0.001), after adjusting for age, BMI, and WHR. CONCLUSIONS: 25(OH) D concentrations are associated with body composition variables especially by BF, independently of seasonal variability. Therefore, body adiposity should be considered when assessing vitamin D requirements in obese patients.

Identification of selective inhibitors of VHSV from biased combinatorial libraries of N,N'-disubstituted 2,5-piperazinediones.

A combinatorial strategy has been used to design and identify inhibitors of viral haemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus of economic importance. Two libraries of N,N'-disubstituted 2,5-piperazinediones (DKP), DKP-I and DKP-II were screened for inhibition of VHSV infectivity. Among the 98 DKP-derivatives (R1-DKP-R2) screened, a novel class of VHSV in vitro inhibitors was identified. Evidences are presented showing that the selected DKP-derivatives cause dose-dependent inhibition of VHSV infectivity in the absence of cellular toxicity. Preliminary characterization of its inhibition mechanism ruled out direct inactivation of the virus (virucidal effect) or interference with early viral replication steps. Furthermore, analysis of infection foci sizes, virus titers, viral protein accumulation and presence of cell free virus derived from VHSV-infected cell cultures in the presence of DKP-derivates suggested that virus assembly/release was impaired leading to a reduced virus spread in cell culture. New DKP-derivatives with a significant higher specific activity need to be developed to start testing its possible practical use but the selected DKP-derivatives described here may contribute to their further development as well as being tools to improve our knowledge on the fish rhabdovirus infection cycle.

Fish transposons and their potential use in aquaculture.

A large part of repetitive DNA of vertebrate genomes have been identified as transposon elements (TEs) or mobile sequences. Although TEs detected to date in most vertebrates are inactivated, active TEs have been found in fish and a salmonid TE has been successfully reactivated by molecular genetic manipulation from inactive genomic copies (Sleeping Beauty, SB). Progress in the understanding of the dynamics, control and evolution of fish TEs will allow the insertion of selected sequences into the fish genomes of germ cells to obtain transgenics or to identify genes important for growth and/or of somatic cells to improve DNA vaccination. Expectations are high for new possible applications to fish of this well developed technology for mammals. Here, we review the present state of knowledge of inactive and active fish TEs and briefly discuss how their possible future applications might be used to improve fish production in aquaculture.

Development of new therapeutical/adjuvant molecules by pepscan mapping of autophagy and IFN inducing determinants of rhabdoviral G proteins.

Surface glycoproteins of enveloped virus are potent elicitors of both innate and adaptive host immune responses. Therefore, the identification of viral glycoprotein determinants directly implicated in the induction of these responses might be of special interest for designing new therapeutical/adjuvant molecules. In this work we review the contribution of the "pepscan" approach to the screening of viral functions in the sequence of glycoprotein G (gpG) of the fish rhabdovirus of viral hemorrhagic septicemia (VHSV). Among others, by scanning gpG peptides, it has been possible to identify and validate minimal determinants for gpG directly implicated in initiating the fish type I Interferon-associated immune responses as well as in the antiviral autophagy program. Further fine-tunning of the identified peptides in the gpG of VHSV has allowed designing novel adjuvants that decrease DNA vaccine requirements and identify possible innovative antiviral molecules. In addition, these results have also contributed to improve our knowledge on how to stimulate the fish immune system.

An in vitro method to obtain T-lymphocyte-like cells from the trout.

We describe a methodology to obtain from the trout T-lymphocyte-like cell cultures showing in vitro antigen-dependent cell (ADC) proliferation. ADC cultures were developed from each of 3 outbred rainbow trout that survived two consecutive viral haemorrhagic septicaemia virus (VHSV) infections (an important salmonid fish disease in Europe). As stimulating antigen, we used a yeast recombinant form of the glycoprotein G (G4) of VHSV. No similar ADC cultures could be obtained from non-infected control trout kidney cells incubated in the presence of G4, confirming previously published observations. For long-term culture, and to overcome the absence of inbred trout populations, autologous haematopoietic adherent (Ad) cells were obtained and maintained from each individual trout kidney and periodically used as viral antigen-presenting cells (called G4-pulsed Ad cells) to autologous ADC cultures. The methodology to obtain trout ADC lines constitutes a new tool that would facilitate further studies on the comparative and developmental immunology of lower vertebrates. It could also be applied to research on fish vaccination and viral antigen-presentation mechanisms.

Structure, binding and neutralization of VHSV with synthetic peptides.

The phosphatidylserine binding region p2 of VHSV was characterized and was shown to be involved with fusion. Synthetic peptides corresponding to this region interact with phospholipids by penetrating into the membrane and changing to a beta sheet configuration. Computer modeling of this region shows the possible ways by which the interaction with the membranes can succeed. Inhibitory peptides are presently being sought by studying possible interactions within heptad repeats located in other regions of the G protein of VHSV. The heptad repeat region that includes the phosphatidylserine binding domain p2 has been cloned and preliminary experiments show that under certain conditions, peptides from this region can inhibit VHSV infectivity.

Enhancement of fish mortality by rhabdovirus infection after immunization with a viral nucleoprotein peptide.

A similar sequence to a mouse immunodominant CTL peptide (SYVLQGN, single-letter amino acid code, conserved amino acids underlined) identified in the nucleoproteins of several strains of vesicular stomatitis virus (VSV) (37) was found in the nucleoproteins of viral hemorrhagic septicemia virus (VHSV) of salmonid fish (GYVYQGL in VHSV 07.71 and GYVYQGS in VHSV Makah) and not in the nucleoproteins of other rhabdoviruses. The in vivo immunization of fingerling salmonid fish (rainbow trout Onchorynchus mykiss, W) with this VHSV peptide and their subsequent challenge with VHSV resulted in the enhancement rather than in the reduction of fingerling trout mortality. Possible implications for the development of subunit vaccines against VHSV are discussed.

Susceptibility of trout kidney macrophages to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) lysed the macrophages from rainbow trout kidney cultures either isolated by plastic adherence or stimulated with purified glycoprotein G from VHSV. The trout macrophages supported the replication of VHSV as tested by cell culture and by sandwich ELISA of the supernatants from infected cultures. VHSV-infected macrophages showed a decrease in both acridine-orange fluorescence and average size. Immunofluorescence studies with flow cytometry showed positive membrane staining with monoclonal antibodies (MAbs) anti-N and anti-G VHSV. These findings open the possibility of using trout macrophages as presenting cells to study the possible existence of helper or cytotoxic epitopes relevant to the protection of trout against VHSV.

Stimulation of adherent cells by addition of purified proteins of viral hemorrhagic septicemia virus to trout kidney cell cultures.

Purified proteins of the virus causing viral hemorrhagic septicemia in the trout were added to cultures on semisolid medium of leukocytes obtained from either healthy or immunized rainbow trout. Adherent cells were specifically stimulated by the glycoprotein of the viral spikes and, to a lesser extent, by the nucleoproteins. In contrast, a specific memory response was associated more with the nucleoproteins than with the glycoprotein when leukocytes from trout immunized with the virus were employed. These results suggest the necessity of employing both proteins in subunit vaccination trials and the possibility of using this assay to select the proper epitopes for genetically engineered proteins during subunit vaccine development.

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Phosphatidylserine binding to solid-phase rhabdoviral peptides: a new method to study phospholipid/viral protein interactions.

A new method is described for the study of phosphatidylserine binding to rhabdoviral peptides by using solid-phase assays. This new assay could probably be extended to study the interactions between host membrane phospholipid and viral proteins in other viruses. By using labeled and hydrated phosphatidylserine (PS), PS-binding to solid-phase 15-mer peptides (pepscan) could map putative phospholipid-binding regions of the glycoprotein G of viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. The major PS-binding region of 27 aa (aa82-109, p2) did not only bind PS, but also phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Extraction of the PS bound to solid-phase p2 by a variety of chemical compounds and competition experiments with several phospholipid-related compounds showed that PS-Binding to p2 was dependent on not only hydrophobic, but also ionic interactions, as suggested by prior work on phospholipid interactions in other rhabdoviruses. Saturation/competition experiments with labeled and cold PS, PE and PC also showed that the reaction probably takes place between high molecular weight aggregates of hydrated phospholipids and several molecules of solid-phase p2. This assay has been used previously to detect hydrophobic amino acid heptad-repeats in rhabdoviruses and when anti-p2 antibodies to VHSV were obtained they were capable of inhibiting VHSV-induced cell to cell fusion.

Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody.

An enzyme-immunohistochemical procedure was employed to facilitate neutralization/diagnostic tests for viral haemorrhagic septicaemia virus (VHSV), a significant pathogen in trout farms throughout Europe. The method described can be used for trout or mice antibodies; increases speed (1 day), simplicity, and minimizes the use of reagents compared to other neutralization assays. Furthermore, the test requires a minimum handling of the cell cultures under sterile conditions, decreasing frequent contamination due to the non-sterile conditions of the fish pathological samples. Foci of 5-20 infected epithelioma papillosum carp (EPC) cells are detected and counted with an inverted microscope in under 16 h after infection of EPC monolayers using a high titre anti-N VHSV monoclonal antibody (MAb) 2C9. MAb 2C9 recognizes different viral haemorrhagic septicaemia virus serotypes and VHSV isolates from different host species (trout, salmon and barbel) and Spanish geographical locations. The high titre and specificity of MAb 2C9 favour its conjugation to peroxidase and also make it possible to use in direct immunoperoxidase staining of the VHSV infected EPC monolayers. This neutralization/immunoperoxidase assay should improve diagnostics that use currently agarose or methylcellulose plaque reduction neutralization assays.