Source: OREGON STATE UNIVERSITY submitted to
USES OF BACULOVIRUS VECTORS FOR THE DEVELOPMENT OF FISH VACCINES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0186478
Grant No.
2001-35204-10116
Project No.
ORE00311
Proposal No.
2000-02106
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 15, 2000
Project End Date
Dec 31, 2005
Grant Year
2001
Project Director
Leisy, D. J.
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
MICROBIOLOGY
Non Technical Summary
The infectious hematopoietic necrosis virus (IHNV) is a serious pathogen of salmonid fishes, especially when reared under hatchery conditions. A DNA vaccine that gives effective protection against IHNV when injected into rainbow trout fry has been developed in Dr. Leong's laboratory. However, injection vaccination is not practical on a large scale, and a more efficient means of delivery of this vaccine needs to be developed, such as oral or immersion administration. We have found that insect baculoviruses can be exploited for the delivery of genes to cultured fish cells, and thus have potential for the delivery of DNA vaccines to fish in vivo. Our strategy is to determine a set of optimized conditions for baculovirus delivery of genes to fish, and use this knowledge to design baculovirus vectors and vaccination protocols that can be used to protect rainbow trout against IHNV disease.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3073711110110%
3113711109010%
3113711110110%
3113714109010%
3113714110110%
3114030109010%
3114030110120%
3153711109010%
3153711110110%
Goals / Objectives
a) Construct prototype baculovirus vaccines that display the IHNV major surface glycoprotein (G-protein) on their surfaces and/or express IHNV-G after transduction into fish cells. b) Optimize conditions for the transduction and expression of baculovirus vectored reporter genes in vivo in two species of fish, tilapia, and rainbow trout. Conditions that will be varied include: temperature, the addition of supplements to the water, and the use of different promoters to drive reporter gene expression. Fish will be exposed to baculovirus vectors by three routes of administration: feeding, immersion and injection. The fate of the baculovirus DNA in various tissues will be ascertained and existing formulation technology will be exploited, if necessary, to optimize oral and/or immersion delivery. c) Viral challenge assays will be performed to evaluate the ability of baculovirus vaccines to protect fish from IHNV. d) The immune responses of fish exposed to vaccine baculoviruses will be monitored.
Project Methods
Recombinant baculoviruses that display the IHNV-G protein on their surface (IHNV-G expressed from the polyhedrin promoter in infected insect cells is predicted to result in viral membrane incorporation of IHNV-G) or express the the IHNV-G gene in transduced fish cells (The IHNV-G gene will be expressed from a promoter active in fish cells) will be constructed and tested as vaccine vectors. Transduced expression of IHNV-G in whole fish is expected to lead to an immune response similar to that induced with the IHNV-G-expressing DNA vaccine. IHNV-G displayed on the surface of the vaccine baculoviruses could also stimulate an immune response and in addition may mediate viral-cell membrane fusion, augmenting the adsorption of the baculovirus by the cell. A dual promoter arrangement will be used to construct baculoviruses that express IHNV-G both on their surfaces and after transduction of fish cells. To facilitate the analysis of foreign gene expression in baculovirus transduced fish cells, baculoviruses that express an easily assayable reporter gene in transduced fish cells will be constructed. To test for transduction in vivo, fish that have been exposed to baculovirus vectors will be fixed and in situ assays for either reporter gene activity or for IHNV-G expression (using alkaline phosphatase immunohistochemical staining) will be performed. These experiments will allow us to optimize conditions (e.g. temperature, supplements, promoter used to drive foreign gene expression, mode of administration, etc.) for the transduction and expression of foreign genes in fish. In vivo lethal challenge assays will be conducted on juvenile rainbow trout in order to assess the protective effects of the various baculovirus vaccines against IHNV infection. Thirty days post-vaccination, replicate groups of fish will be challenged by immersion in water containing various concentrations of IHNV, and compared with unchallenged control groups. Dead fish will be scored and removed daily, and the level of IHNV infection will be determined by plaque assay. Parallel experiments will also be conducted in which IHNV titers will be monitored in surviving fish. The humoral immune response in vaccinated and control fish will be determined by ELISA assays for IHNV-specific immunoglobulin (Ig). Previous experiments from our laboratory have shown that interferon inducers, such as poly I:C, stimulate rainbow trout Mx gene expression. To monitor for the induction of non-specific immune modulators, immunized fish will be assayed for the expression of Mx genes at both the mRNA and protein levels.

Progress 12/15/00 to 12/31/05

Outputs
The long-range goal of this project was to develop commercially viable vaccines for disease control in aquaculture. Previous studies had shown that injection of a DNA vaccine encoding the G protein from infectious hematopoietic necrosis virus (IHNV) provided very effective protection of rainbow trout and other salmonid species against IHNV infection. However, in order for this vaccine to be of practical use a more efficient means of administration such as oral or immersion delivery is required. Based on our initial findings that baculoviruses are capable of delivering genes to a variety of fish cell lines, we initiated a project to determine if baculovirus vectors could be used for delivering genetic vaccines to fish by either of these preferred delivery methods. Recombinant baculoviruses designed to 1) display the IHNV-G protein on their surface, 2) express G-protein after transfection into fish cells, and 3) both display the IHNV-G protein on their virions and express G-protein after transfection into fish cells were tested for their abilities to carry out these functions. Western blot experiments using an anti-IHNV-G monocolonal antibody demonstrated that IHNV-G protein was produced by the 1st and 3rd construct, but that the expressed G-protein was not incorporated into virions, as was expected based on baculovirus expression studies with the G -protein from vesicular stomatitis virus. We also found that G-protein expression by the 2nd construct in transfected fish cells was only detectable when a very high MOI was used at temperatures well above those tolerated by rainbow trout. However, we did find that rainbow trout injected with these recombinant viruses, or with control baculoviruses that do not encode the IHNV-G protein, were protected when challenged with IHNV 6 days post-injection, but no protection was evident when the challenge occurred at 60 days post-injection, indicating the induction of an innate immune response. Gavage administration of baculoviruses did not provide protection, however anal administration provided a low but statistically significant level of protection. The mode of action of the protective innate immune response is uncertain, as induction of Mx, an indicator of an interferon-mediated response, was not detected in Western blots. We found that baculovirus rapidly lost biological activity (plaque forming ability on Sf9 cells) when exposed to fluids extracted from the lumens of the stomach or small intestine, but retained activity when subjected to fluids extracted from the large intestine. Based on these observations we reasoned that it might be possible to deliver genetic vaccines orally by encasing them in virus-like-particles (VLPs) derived from an orally-transmitted virus. Preliminary experiments in which baculovirus-produced VLPs derived from hepatitis E virus were delivered by injection, gavage, and anal intubation provided no indication that the empty VLPs induce an innate immune response, as was observed with baculoviruses. However, further studies are warranted to determine if either an innate or adaptive immune response would be induced by VLPs harboring a genetic vaccine.

Impacts
Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen that can cause losses as high as 90% in trout farms and hatcheries. Outbreaks usually require the complete destruction of all fish in a hatchery in order to control the spread of the virus. In the Columbia River Basin IHNV has caused the destruction of an estimated 300 million fish since 1985, not counting the mortalities in private rainbow trout farms on the Columbia River tributaries. A very effective DNA vaccine against IHNV has been developed, but since it must be administered by injection of individual fish, its use in hatcheries and trout farms is limited. Our efforts to develop baculoviruses for immersion or oral delivery of DNA vaccines to fish could have a significant impact on hatcheries, the trout farm industry, and on consumers. To get an idea of the potential economic benefits, consider that U.S. trout farmers produced $65.2 million worth of trout in 2002. Total losses of all trout intended for sale were 35.0 million fish with 83% of the loss due to disease (mostly viral infections). If commercially viable vaccines were to become available, a high percentage of the 35 million fish now lost might be salvageable.

Publications

  • Leisy, D. J. Development of oral delivery vehicles for fish vaccines. Center for Fish Disease Research Colloquium. Oregon State University, April 1, 2005.
  • Leisy, D. J., M. Alonso, and J.-A. C. Leong. IHNV vaccine development. Center for Fish Disease Research Colloquium. Oregon State University March 15, 2004.
  • Leisy, D. J., T. D. Lewis, J.-A. C. Leong, and G. F. Rohrmann. 2003. Transduction of cultured fish cells with recombinant baculoviruses. J. Gen. Virol. 84:1173-8.
  • Leisy, D. J. & J.A.-C. Leong. Construction and evaluation of recombinant baculoviruses as vaccines against infectious hematopoietic necrosis virus. American Society for Virology 22nd Annual Meeting. University of California, Davis. July 12-16, 2003.
  • Leisy, D. J. Baculovirus-based vaccines for use in aquaculture. Aquaculture/Fish Health Workshop. Oregon State University Hatfield Marine Science Center, Newport, OR. April 3, 2002.
  • Leong, J. C., M. Alonso, D. Leisy, T. Lewis, B. Robertsen, B. Simon, C. B. Song, & E. Thomann. Genetic Vaccines For Aquaculture. Biotechnology-Aquaculture Interface: The Site of Maximum Impact Workshop. Shepherdstown, West Virginia, Mar. 5-7, 2001. Sponsored by ARS-USDA, Beltsville, Maryland and The Oceanic Institute, Waimanalo, Hawaii.


Progress 01/01/04 to 12/31/04

Outputs
Our long-range goal is to develop commercially viable vaccines for disease control in aquaculture. Previous studies have shown that injection of a DNA vaccine encoding the G protein from infectious hematopoietic necrosis virus (IHNV) provides very effective protection of rainbow trout and other salmonid species against IHNV infection. However, in order for this vaccine to be of practical use a more efficient means of administration, such as oral or immersion delivery must be developed. Based on our initial findings that baculoviruses are capable of delivering genes to a variety of fish cell lines, we initiated experiments to determine if baculovirus vectors can be developed that are capable of delivering genetic vaccines to fish by either of these preferred delivery methods. Recombinant baculoviruses designed to: 1) display the IHNV-G protein on their surface, 2) express G-protein after transfection into fish cells, and 3) both display the IHNV-G protein on their virions and express G-protein after transfection into fish cells were tested for their abilities to carry out these functions. Western blot experiments using the anti-IHNV-G monoclonal antibody 136J showed that expression of IHNV G-protein in baculovirus-infected insect cells did not result in measurable incorporation of G-protein into baculovirus virions, as was expected based on studies with vesicular stomatitis virus G-protein. Western blots showed that baculoviruses designed to induce expression of IHNV-G in transfected fish cells have the ability to do so, however a positive signal was only observed when the transfected cells were maintained at 30 degrees C and an extremely high multiplicity of infection (moi=900) was used. Infection at moi =90 did not give a positive signal at any of the three temperatures tested (17, 21 and 30 degrees C). Rainbow trout injected with these recombinant viruses, or with control baculoviruses that do not encode the IHNV-G protein, were protected when challenged with IHNV six days post treatment. No protection was evident, however, when the challenge occurred 60 days post treatment. Gavage administration of baculoviruses was ineffective at protecting rainbow trout against IHNV infection. Although our results to date are negative with regard to baculovirus delivery of antigenic and DNA vaccines to fish, we have demonstrated that baculovirus-injected rainbow trout mount a strong, nonspecific immune response capable of protecting them against IHNV infection.

Impacts
Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen that can cause losses as high as 90% in trout farms and hatcheries. Outbreaks usually require the complete destruction of all fish in a hatchery in order to control the spread of the virus. In the Columbia River Basin IHNV has caused the destruction of an estimated 300 million fish since 1985, not counting the mortalities in private rainbow trout farms on the Columbia River tributaries. A very effective DNA vaccine against IHNV has been developed, but since it must be administered by injection of individual fish, its use in hatcheries and trout farms is limited. Our efforts to develop baculoviruses for immersion or oral delivery of DNA vaccines to fish could have a significant impact on hatcheries, the trout farm industry, and on consumers. To get an idea of the potential economic benefits, consider that U.S. trout farmers produced $65.2 million worth of trout in 2002. Total losses of all trout intended for sale were 35.0 million fish with 83% of the loss due to disease (mostly viral infections). If commercially viable vaccines were to become available, a high percentage of the 35 million fish now lost might be salvageable.

Publications

  • Leisy, D. J., M. Alonso, and J.-A. C. Leong. IHNV vaccine development. Center for Fish Disease Research Colloquium. Oregon State University March 15, 2004.


Progress 01/01/03 to 12/31/03

Outputs
Our long-range goal is to develop commercially viable vaccines for disease control in aquaculture. Previous studies have shown that injection of a DNA vaccine encoding the G protein from infectious hematopoietic necrosis virus (IHNV) provides very effective protection of rainbow trout and other salmonid species against IHNV infection. However, in order for this vaccine to be of practical use a more efficient means of administration, such as oral or immersion delivery, must be developed. Based on our initial findings that baculoviruses are capable of delivering genes to a variety of fish cell lines, we initiated experiments to determine if baculovirus vectors can be developed that are capable of delivering genetic vaccines to fish by either of these preferred delivery methods. We have made recombinant baculoviruses that carry the IHNV-G gene downstream from: 1) a promoter expected to be active in infected insect cells which should lead to the expression of the G protein on the baculovirus surface, 2) a promoter expected to be active in transfected fish cells, allowing expression of G in these cells, and 3) a dual promoter arrangement designed to allow IHNV-G expression in both insect and fish cells. During this reporting period these recombinant baculoviruses were tested for their ability to immunize rainbow trout after injection. When fish were challenged at 6 days post vaccination, these baculovirus constructs, and another control baculovirus that did not contain the G gene, protected fish as effectively as injected IHNV-G expressing plasmids. Our results demonstrate that baculoviruses are effective at protecting rainbow trout against infection with IHNV when delivered by injection. Since the response is rapid (occurring within 6 days) and is elicited by non-recombinant baculoviruses, it is likely due to activation of a non-specific viral defense mechanism in the fish. We are currently conducting longer-term experiments to determine if injected recombinant baculoviruses can induce the production of IHNV-neutralizing antibodies and long-term specific immunity to IHNV. These tests will be followed by studies to develop systems for immersion and/or oral delivery of the baculovirus vaccines.

Impacts
Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen that can cause losses as high as 90% in trout farms and hatcheries. Outbreaks usually require the complete destruction of all fish in a hatchery in order to control the spread of the virus. In the Columbia River Basin IHNV has caused the destruction of an estimated 300 million fish since 1985, not counting the mortalities in private rainbow trout farms on the Columbia River tributaries. A very effective DNA vaccine against IHNV has been developed, but since it must be administered by injection of individual fish, its use in hatcheries and trout farms is limited. Our efforts to develop baculoviruses for immersion or oral delivery of DNA vaccines to fish could have a significant impact on hatcheries, the trout farm industry, and on consumers. To get an idea of the potential economic benefits, consider that U.S. trout farmers produced $65.2 million worth of trout in 2002. Total losses of all trout intended for sale were 35.0 million fish with 83% of the loss due to disease (mostly viral infections). If commercially viable vaccines were to become available, a high percentage of the 35 million fish now lost might be salvageable.

Publications

  • Leisy, D. J., T. D. Lewis, J.-A. C. Leong, and G. F. Rohrmann. 2003. Transduction of cultured fish cells with recombinant baculoviruses. J. Gen. Virol. 84:1173-8.
  • Leisy, D. J. & J.A.-C. Leong. Construction and evaluation of recombinant baculoviruses as vaccines against infectious hematopoietic necrosis virus. American Society for Virology 22nd Annual Meeting. University of California, Davis. July 12-16, 2003.


Progress 01/01/02 to 12/31/02

Outputs
In this study we are investigating the feasibility of using recombinant baculoviruses as fish vaccines. Our earlier studies provided evidence that recombinant baculoviruses are capable of delivering reporter genes to several different fish cell lines. If the reporter genes are cloned so that they are downstream from a promoter that is active in the fish cells, they become expressed. We made a number of different recombinant baculoviruses that are expected to express the IHNV-G gene either on the surface of the virions (via expression from a promoter active in the insect cell line used to propagate the baculovirus), or after transduction into fish cells. To test the integrity of baculovirus transfer plasmids used as intermediates in the construction of IHNV-G expressing baculoviruses, we injected fish with these plasmids and conducted two types of assays: 1) serum neutralization assays in which the ability of serum from immunized rainbow trout was examined for its ability to inhibit plaque formation by IHNV in vitro, and 2) IHNV challenge assays in which we examined the ability of plasmid-injected fish to resist infection by IHNV. We found that fish injected with plasmids containing the IHNV-G gene downstream from either the CMV or CAG promoter produced IHNV-neutralizing sera. In challenge assays these same plasmids rendered fish 100% immune (or nearly so) to viral challenge. However, fish injected with constructs containing the IHNV gene downstream of the baculovirus polyhedrin promoter did not produce neutralizing sera nor did they exhibit immunity to IHNV infection. This does not mean, however, that a recombinant baculovirus containing the IHNV-G gene under control of the polyhedrin promoter would not be immunogenic, since it could stimulate the fish immune system via IHNV-G antigen expressed on its surface. Detection of IHNV-G on these virions by Western blots has so far been hampered by the low specificity of the anti-IHNV-G antibodies that we have available. We have conducted one preliminary challenge assay of fish exposed via emersion to a recombinant baculovirus (100,000,000 pfu/ml) containing the IHNV-G gene downstream of both the CAG promoter and the baculovirus p10 promoter. There were no differences in susceptibility to IHNV infection in fish exposed to this baculovirus, a wild type control baculovirus and a mock-vaccinated group. We are now preparing to examine whether recombinant baculoviruses delivered via either injection or ingestion can effectively immunize fish.

Impacts
Recent studies have demonstrated superior efficacy of DNA based vaccines for immunization of fish compared to more traditional vaccines such as inactivated or attenuated vaccines, however, the practical application of such DNA-based vaccines is limited because they have only been found to be effective when delivered by injection, gene gun, scarification or by the application of short-pulse ultrasound, and not by ingestion or simple bath immersion. We are interested in testing the possibility of using transducing baculoviruses to deliver DNA modules to fish during immersion, which would be a preferred route of administration in hatchery situations in which large numbers of fry need to be vaccinated. The results of our study indicate that baculovirus vectored foreign genes can be expressed in a variety of fish cell lines, and can be modified to allow increased expression of foreign genes, providing incentive for further investigations on the development of baculovirus-based vaccines for use in fish.

Publications

  • Leisy, D. J. Baculovirus-based vaccines for use in aquaculture. Aquaculture/Fish Health Workshop. Oregon State University Hatfield Marine Science Center, Newport, OR. April 3, 2002.


Progress 01/01/01 to 12/31/01

Outputs
In this study we are investigating the feasibility of using recombinant baculoviuses as fish vaccines. Preliminary studies indicated that we could detect transfer and expression of the b-galactosidase gene in four of five fish cell lines transduced with recombinant baculoviruses. Using the EPC cell line for further studies we showed a positive correlation between MOI and expression, that expression could be augmented approximately 5-6 fold by supplying sodium butyrate to the culture medium, that recombinant baculoviruses displaying a heterologous viral membrane protein (the vesicular stomatitis virus G-protein) had about a 15-fold higher level of expression, and that expression levels were much higher at 28 degrees centigrade than at 21 or 17 degrees. We have now used temperature-shift experiments to demonstrate that temperatures in the range tested (17-28oC) had no affect on the ability of the cells to adsorb virus, but only affect the level of gene expression after adsorption. The observed temperature effects are probably due to reduced transcription of the reporter gene at the lower temperatures. Since fish are typically maintained at lower temperature, it may be necessary to use a promoter that retains high levels of transcription at lower temperatures. Several different promoters were tested for their ability to drive expression at the lower temperatures and it was found that the CAG promoter used in our early constructs was least effective, and that the CMV promoter was most effective. We have made several new constructs using the CMV promoter to drive either the luciferase reporter gene, or the IHNV-G gene vaccine module. Luciferase reporter gene constructs include: 1) A baculovirus vector that displays the IHNV-G protein, 2) A control baculovirus vector that does not display a heterologous membrane protein. For comparison a third construct displaying the VSV-G protein is under construction. These constructs will be used to determine if these heterologous membrane proteins will lead to enhanced luciferase gene expression in baculovirus transduced cell lines. We have made three new constructs containing the IHNV-G gene. Construct 1 has the IHNV-G gene under the control of both the polyhedrin (pY) and the CMV promoter and is expected to display IHNV-G protein on the virions, as well as express IHNV in transduced cells. Construct 2 has the IHNV-G gene only under the control of the pY promoter and should display IHNV-G protein on the virions only. Construct 3 has the IHNV-G gene only under the control of the CMV promoter and is expected to express IHNV only in transduced cells. These vectors will be used to vaccinate rainbow trout, and tested by challenging the vaccinated fish with IHNV. If protection is observed, comparison of the vaccine efficiency of these three constructs should allow us to determine whether vaccination is occurring: a) as a result of the IHNV-G antigen on the virion surface, b) as a result of IHNV-G gene expression in transduced cells, or c) a combination of these two effects.

Impacts
Recent studies have demonstrated superior efficacy of DNA based vaccines for immunization of fish compared to more traditional vaccines such as inactivated or attenuated vaccines, however, the practical application of such DNA-based vaccines is limited because they have only been found to be effective when delivered by injection, gene gun, scarification or by the application of short-pulse ultrasound, and not by ingestion or simple bath immersion. We are interested in testing the possibility of using transducing baculoviruses to deliver DNA modules to fish during immersion, which would be a preferred route of administration in hatchery situations in which large numbers of fry need to be vaccinated. The results of our study indicate that baculovirus vectored foreign genes can be expressed in a variety of fish cell lines, and can be modified to allow increased expression of foreign genes, providing incentive for further investigations on the development of baculovirus-based vaccines for use in fish.

Publications

  • No publications reported this period