Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination

Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 microg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene (pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immune-related genes, e.g. MHC class Ialpha, IIalpha, IIbeta, TCR-alpha, beta1, beta2 and delta, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR beta1 and beta2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization.     

类泛素蛋白ISG15及其共价酶修饰系统与肿瘤

干扰素刺激基因15(interferon-stimulated gene 15,ISG15)编码的ISG15蛋白是最早发现的类泛素修饰蛋白。不仅干扰素和病毒感染能诱导该基因的表达,一些抗肿瘤药物也能刺激ISG15的表达。近年来,ISG15及其修饰系统与肿瘤的关系备受关注。研究证实,ISG15的激活酶E1即UBE1L与某些肿瘤的抑制有关,而其他一些成分如ISG15解聚酶UBP43则与肿瘤发生发展有关。此外,ISG15高表达与肿瘤的转移有关,ISG15对化疗药物的敏感性也有影响。本文较为全面地阐述ISG15及其修饰系统在肿瘤抑制或发生发展中的生物学作用,这将增强对ISG15与肿瘤关系的基础性认识,并为发展新的肿瘤靶向性治疗提供理论依据。     

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.     

mHERC6 is the essential ISG15 E3 ligase in the murine system

Posttranslational protein modification by ubiquitin and ubiquitin-like modifiers (UBLs) is mediated by a hierarchical cascade of conjugating enzymes and affects multiple biological processes within the cell. Interferon-stimulated gene 15 (ISG15) is an UBL, which is strongly induced by type I Interferon and ISG15 modification was shown to play an essential role in antiviral defense. While hHERC5 is the major E3 ligase for ISG15 modification in humans, ISGylation in the murine systems at the level of E3 ligases was weakly characterized as rodent genomes lack a direct homologue of hHERC5. Here, we show that mHERC6 is strongly induced by different pathogen-associated molecular patterns (PAMPs) in a type I Interferon receptor (IFNAR1) dependent manner. We demonstrate that mHERC6 is essential for endogenous murine ISGylation and thus represents the dominant ISG15 E3 ligase in mice. In contrast to its human homologue, mHERC6 is also capable to mediate conjugation of human ISG15.     

Approaches towards DNA Vaccination against a Skin Ciliate Parasite in Fish

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.     

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.     

Crystal structure of ISG54 reveals a novel RNA binding structure and potential functional mechanisms

Interferon-stimulated gene 56 (ISG56) family members play important roles in blocking viral replication and regulating cellular functions, however, their underlying molecular mechanisms are largely unclear. Here, we present the crystal structure of ISG54, an ISG56 family protein with a novel RNA-binding structure. The structure shows that ISG54 monomers have 9 tetratricopeptide repeat-like motifs and associate to form domain-swapped dimers. The C-terminal part folds into a super-helical structure and has an extensively positively-charged nucleotide-binding channel on its inner surface. EMSA results show that ISG54 binds specifically to some RNAs, such as adenylate uridylate (AU)-rich RNAs, with or without 5′ triphosphorylation. Mutagenesis and functional studies show that this RNA-binding ability is important to its antiviral activity. Our results suggest a new mechanism underlying the antiviral activity of this interferon-inducible gene 56 family member.     

Immunogenicity, retention and protective effects of the protein derivatives of formalin-inactivated red seabream iridovirus (RSIV) vaccine in red seabream, Pagrus major

A formalin-inactivated virus was previously found to be efficient in protecting fish against challenge with red seabream iridovirus (RSIV), a DNA virus belonging to the Iridoviridae family. In the present study, we determined the amount of the virus in the vaccine in terms of the number of copies of the gene for the major capsid protein (MCP) gene by quantitative real-time PCR and examined the longevity and types of immune response generated after intramuscular vaccination. We also tested whether the protein components of the vaccine are able to mount a protective immune response in fish. The vaccine contained 10(7) MCP copies per microliter of vaccine, and was detected in blood, kidney and spleen of vaccinated fish up to 15 days post-vaccination. Fish vaccinated with either the intact formalin-inactivated vaccine or its protein derivatives had increased serum neutralization antibodies and enhanced expression of MHC class I, although the kinetics of expression varied among groups. However, only those vaccinated with the intact vaccine survived the virus challenge, and this indicates that serum neutralization antibodies have scarce role in protecting the fish against RSIV. We hypothesize that the cell-mediated immunity, particularly the MHC class I pathway is responsible for such protection.     

Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.     

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.     

Modulation of the early immune response against viruses by a teleostean interferon regulatory factor-1 (IRF-1)

We investigated the role of a teleostean interferon regulatory factor-1 (IRF-1) in the regulation of the fish immune system using Japanese flounder, Paralichthys olivaceus, as a model. Fish were intramuscularly vaccinated with a recombinant plasmid expressing the Japanese flounder IRF-1 (JF IRF-1) under the control of the cytomegalovirus immediate/early enhancer (CMV) promoter and were sampled at different days post-immunization. Peripheral blood leukocytes (PBLs) obtained from the JF IRF-1-vaccinated fish during the early stages post-vaccination had significantly elevated levels of nitric oxide (NO) and higher acid phosphatase (AP) activity compared with the control groups. Moreover, supernatants of PBLs obtained from the IRF-1-vaccinated fish contained cytokine-like substances as shown by their protective effect against hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV) in two cell lines, hirame natural embryo (HINAE) cell line and epithelial papillosum of cyprini (EPC) cell line. Relative expression of an anti-viral gene, Mx was highest at the 7th day post-vaccination. Co-injection of JF IRF-1 with a DNA vaccine encoding the major capsid protein (MCP) gene of red seabream iridovirus (RSIV) resulted in elevated serum neutralization antibodies but was not significantly different from that in the fish vaccinated with the DNA vaccine alone. These results suggest that the JF IRF-1 modulates the early immune response in fish and is a potential candidate as genetic adjuvant for vaccination.     

The Antiviral Activities of ISG15

Post-translational protein modification is an important strategy for the regulation of the cell proteome independent of the need for new gene expression. Ubiquitin and ubiquitin-like modifiers mediate the regulation of protein levels, signaling pathways, vesicular trafficking, and many other cellular processes through their covalent conjugation to proteins. Interferon stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I interferon. In addition to conjugating to potentially hundreds of target proteins, ISG15 can be found in an unconjugated form both inside of the cell and released from interferon stimulated cells into the extracellular environment. Due to its robust expression after type I interferon stimulation and the broad panel of proteins that it targets, ISG15 has drawn much attention as a potential regulator of the immune response and has been shown to mediate protection in a number of different viral infection models. Here we will review the current state of the field of ISG15, the viruses against which ISG15 mediates protection, and the mechanisms by which ISG15 exerts antiviral activity.     

类泛素修饰蛋白质ISG15及其修饰酶系的功能

受干扰素诱导表达的干扰素刺激基因15编码蛋白质(ISG15)是第1个被鉴定的类泛素修饰蛋白质.目前已在病毒感染细胞和肿瘤细胞中发现了多种ISG15的作用靶蛋白,提示ISG15可能在免疫调节和肿瘤发生等方面发挥重要作用.本文介绍ISG15的结构与生化特点,探讨ISG15在相关酶系作用下修饰目标蛋白质的机制,总结ISG15及其修饰酶系的抗病毒和抗肿瘤作用及其相关机制.