Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss).

Fragments of the glycoprotein genes of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) were cloned into a bacterial broad-host-range expression vector under the control of the plac promoter. Western blot (immunoblot) analysis with monoclonal antibodies specific to the glycoproteins demonstrated the inducible expression of the fusion proteins in Escherichia coli. Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. It was confirmed that an avirulent strain of A. salmonicida, A440, which contains a deletion in the structural gene for the paracrystalline surface protein array, will provide protective immunity against furunculosis when used as a live attenuated vaccine. The plasmid-encoded viral epitopes were then mobilized into A440 for use as a shuttle system for the expression of fragments of the glycoprotein genes of IHNV and VHSV. Vaccination of rainbow trout with A440 containing the viral epitopes resulted in the development of protective immunity against both VHSV and IHNV. This indicates that the use of cloned fragments of the glycoproteins and the use of A. salmonicida as a shuttle system constitute a feasible approach to fish vaccine development.     

Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

HPV16型E7复制型DNA疫苗诱发的抗肿瘤免疫反应

为了研制有效的疫苗 ,用于HPV16型重度感染和与其感染相关的宫颈癌晚期病人手术后的免疫治疗 ,用复制型DAN疫苗载体 pSCA1,在其CMVIE启动子之下插入修饰的HPV16型E7基因mE7- 3( 2 4G、2 6G、6 7R) ,构建成 pSCA1mE7- 3复制型DNA疫苗。以重组质粒免疫C5 7BL/ 6小鼠 ,检测诱发的特异性CTL活性 ;将免疫后的小鼠用TC - 1肿瘤细胞攻击 ,观察免疫保护效果。实验结果显示 :pSCA1mE7- 3复制型DNA疫苗能诱导小鼠产生针对TC - 1肿瘤细胞的特异性CTL反应 ;复制型DNA疫苗免疫后的小鼠能耐受 1× 10 4 TC - 1细胞的攻击 ,成瘤时间推迟 ,并且成瘤率明显下降 ,部分小鼠得到保护能免受肿瘤攻击。因此pSCA1mE7- 3复制型DNA疫苗可作为HPV16相关肿瘤的癌前病变及中晚期病人术后免疫治疗的候选疫苗。     

Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV

A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.     

DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish

Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.     

Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.     

Specific inhibition of gene expression using a stably integrated,inducible small-interfering-RNA vector

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted β-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated β-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

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 β-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.     

Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon

The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.     

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.     

Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV)

This article reviews some of the recent patents on DNA vaccines against fish viruses, in particular against the novirhabdovirus infectious hematopoitic necrosis virus (IHNV). Although very effective in protecting fish against IHNV, only one DNA vaccine has been approved to date for use in Canada. In Europe and in US, its commercialization is restricted due to safety concerns.     

Analysis of DNA-Vaccinated Fish Reveals Viral Antigen in Muscle, Kidney and Thymus, and Transient Histopathologic Changes

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was used in a systematic study to analyze vaccine tissue distribution, persistence, expression patterns, and histopathologic effects. Vaccine plasmid pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately after intramuscular injection in all tissues analyzed, including blood, but at later time points was found primarily in muscle tissue, where it persisted to 90 days. Glycoprotein expression was detected in muscle, kidney, and thymus tissues, with levels peaking at 14 days and becoming undetectable by 28 days. Histologic examination revealed no vaccine-specific pathologic changes at the standard effective dose of 0.1 μg DNA per fish, but at a high dose of 50 μg an increased inflammatory response was evident. Transient damage associated with needle injection was localized in muscle tissue, but by 90 days after vaccination no damage was detected in any tissue, indicating the vaccine to be safe and well tolerated.     

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.     

Vector potential of the salmon louse Lepeophtheirus salmonis in the transmission of infectious haematopoietic necrosis virus (IHNV)

To better understand the role of vector transmission of aquatic viruses, we established an in vivo virus-parasite challenge specifically to address (1) whether Lepeophtheirus salmonis can acquire infectious haematopoietic necrosis virus (IHNV) after water bath exposure or via parasitizing infected Atlantic salmon Salmo salar and if so, define the duration of this association and (2) whether L. salmonis can transmit IHNV to naive Atlantic salmon and whether this transmission requires attachment to the host. Salmon lice which were water bath-exposed to 1 x 10(5) plaque-forming units (pfu) ml(-1) of IHNV for 1 h acquired the virus (2.1 x 10(4) pfu g(-1)) and remained IHNV-positive for 24 h post exposure. After parasitizing IHNV-infected hosts (viral titer in fish mucus 3.3 x 10(4) pfu ml(-1)) salmon lice acquired IHNV (3.4 x 10(3) pfu g(-1)) and remained virus-positive for 12 h. IHNV-positive salmon lice generated through water bath exposure or after parasitizing infected Atlantic salmon successfully transmitted IHNV, resulting in 76.5 and 86.6% of the exposed Atlantic salmon testing positive for IHNV, respectively. In a second experiment, only salmon lice that became IHNV-positive through water bath exposure transmitted IHNV to 20% of the naive fish, and no virus was transmitted when IHNV-infected salmon lice were cohabitated but restrained from attaching to naive fish. Under laboratory conditions, adult L. salmonis can acquire IHNV and transmit it to naive Atlantic salmon through parasitism. However, the ephemeral association of IHNV with L. salmonis indicates that the salmon louse act as a mechanical rather than a biological vector or reservoir.