Progress 09/15/04 to 09/14/08
Outputs
OUTPUTS: The primary output from this project is our development of a pre-commercial ISAV recombinant protein vaccine prototype. The ISA virus model system used in these studies had been developed previously using the CCBB isolate of ISA virus recovered from infected farmed Atlantic salmon in New Brunswick, Canada. The degree of similarity between the CCBB and ME ISA virus isolates indicated to us that a recombinant vaccine based upon the CCBB ISA virus proteins would likely protect vaccinated fish against ISA virus present in Maine and New Brunswick. The research conducted during our Phase I award revealed the presence of immunoprotective epitopes within the hemagglutinin esterase (HE), the nucleoprotein (NP) and the fusion protein (F) of ISAV. These three proteins continued to be the focus of our Phase II project but we utilized sophisticated bioinformatic tools to identify additional putative immunoprotective epitopes. Three generations of ISAV recombinant protein vaccine prototypes encompassing 38 different chimeric antigens were constructed and tested during the course of the Phase II project. Through an iterative process that built upon the knowledge gained from each preceding step, we selected four immunoprotective epitopes from two ISAV antigens. We hypothesized that their protective properties would be augmented by combining them in tandem with promiscuous T cell epitopes (TCE) and multiple copies of a molecular adjuvant. We predicted that induction of both the innate and adaptive immunity in immunized salmon was required for an ISAV vaccine to be effective and tested this postulate by fusing the ISAV epitopes with either the TCE or the molecular adjuvant or both epitopes. Our efforts culminated in the creation of six heteropolyepitope recombinant proteins. Our next step was to establish the guidelines and procedures for large-scale expression, purification and sterilization of the recombinant protein from the expression host. We refined the purification process to optimize the yield of each recombinant protein and reduce the downstream processing costs. Finally, the safety and efficacy of the vaccines were established through a series of studies that examined the effect of different antigens and antigen combinations, the dose of recombinant antigen, types of adjuvants (water-in-oil, oil-in-water), the challenge model (injected versus cohabitation) and the duration of immunity elicited in fish vaccinated with the recombinant protein vaccines. Throughout the project, we also assessed the susceptibility of ISA convalescent Atlantic salmon to virus re-exposure and their virus carrier status. The results of these studies will have significant implications for fish health management strategies in the field. PARTICIPANTS: PI - Dr. Sharon Clouthier: scientific researcher and research manager for the project; Co-PI - Dr. Eric Anderson: scientific researcher and co-manager for the project; Technician - performed laboratory experiments under the direction of the PI and the co-PI. TARGET AUDIENCES: The Phase II research is relevant to the scientific community and the aquaculture industry. PROJECT MODIFICATIONS: The project took a total of 48 months to complete. At the end of year two and year three, we received one-year no-cost extensions for the project as the length of time required to identify the appropriate antigen for our ISAV recombinant vaccine was more than we had initially anticipated.
Impacts
The overall goal of this project was to develop a vaccine for the control of ISA disease in farmed Atlantic salmon. We have successfully completed the objectives outlined for the project and the results provide the foundation for commercialization of a safe and efficacious ISAV recombinant protein vaccine. An ISAV vaccine will play an important role in the sustainability of the salmon aquaculture industry. Immunity of farmed fish to disease reduces the potential for pathogen bio-amplification and transmission between farmed and wild fish, lowers the cost of production and fulfills market, trade and consumer preferences for wholesome aquaculture products produced without the aid of antimicrobials and chemicals. The first milestone in the development of a recombinant protein vaccine is the identification of immunogenic epitopes within proteins that are considered to play a role in the protective response of the host to the pathogen. A total of 31 different epitopes from six different ISAV proteins were presented as either monoepitope or heteropolyepitope fusion proteins. Through an iterative process, we selected four of these epitopes and fused them in tandem with promiscuous T cell epitopes and a molecular adjuvant. We believe that one of these heteropolyepitope recombinant proteins is the final version of the vaccine we have been working towards for the past four years. The second goal in the development of a recombinant protein vaccine is to establish procedures for large-scale expression and purification of the protein. Conditions were established for the culture of bacteria in liquid media and aerobic growth in flasks or fermentation vessels. Expression of the recombinant protein was tightly controlled and the antigen produced was directed to inclusion bodies, facilitating purification and downstream processing. With this system, greater than 50% of the cellular protein was the recombinant fusion protein. These characteristics made this a very cost effective method for producing large-scale quantities of the recombinant protein. The third critical step in the development of a recombinant protein vaccine is to test whether the vaccine performs adequately in immunized animals. We conducted a total of nine in vivo studies during the Phase II project to test the safety and efficacy of these recombinant protein vaccines in Atlantic salmon. A number of factors that influenced the efficacy of these vaccines were identified including intrinsic properties of the antigen, synergistic or antagonistic interactions between antigens, antigen dose, type of adjuvant and method of challenge. The recombinant vaccines were well tolerated when administered by intraperitoneal injection to Atlantic salmon; no mortalities occurred and there were no overt signs of adverse effects. The most promising vaccine prototype was a third generation heteropolyepitope recombinant protein vaccine prototype that provided a relative percent survival of 70 in immunized salmon challenged by cohabitation 5 months after vaccination. As a commercial product, this vaccine will provide an alternative fish health management strategy to the salmon aquaculture industry.
Publications
- Clouthier, S., B. Glebe, W. Young-Lai and E. Anderson (2008) Infectious salmon anemia virus (ISAV) vaccines. American Fisheries Society Fish Health Section Annual Meeting, Charlottetown, PEI, Canada.
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Maine BioTek, Inc. received USDA SBIR Phase II funds for the proposal entitled "Recombinant ISA virus vaccine". The proposal was directed to research topic "Aquaculture (8.7)" under the subtopic "Integrated Aquatic Animal Health Management". The overall goal of the project is to develop a recombinant vaccine for the control of infectious salmon anemia (ISA) disease of farmed Atlantic salmon. The work addresses five primary topics related to vaccine performance: vaccine (1) construction, (2) formulation, (3) potency, (4) safety and (5) efficacy. The results will serve as the scientific basis for Phase III work involving the submission of a dossier for regulatory approval and subsequent commercialization of the vaccine in conjunction with our strategic partner. The objectives of the Phase II project are to examine the effect of different vaccine doses, vaccination regimes, combinations and arrangements of antigens and immunostimulatory epitopes and adjuvant formulation on
vaccine efficacy, to determine the kinetics and duration of the protective immune response and to establish the level of cross-strain protection elicited by the vaccine(s). The progress within each of the tasks required to complete the Phase II technical objectives is summarized as follows: A)Construction of recombinant clones - completed (Generation 1: 4 constructs; Generation 2: 34 constructs; Generation 3: 6 constructs); B) Expression & purification of recombinant proteins - completed; C) Vaccine formulation, sterility, safety & stability - completed; D) Vaccine performance: 1) Dose response - completed; 2) Combinations of recombinant proteins - completed (study 1 & 2 completed; additional study 3 ongoing); 3) Formulation with different adjuvants - completed; 4) Duration of immunity - ongoing.
PARTICIPANTS: PI - Dr. Sharon Clouthier: scientific researcher and research manager for the project; Co-PI - Dr. Eric Anderson: scientific researcher and co-manager for the project; Technician: performs laboratory experiments under the direction of the PI and the co-PI.
PROJECT MODIFICATIONS: We have requested a one-year no-cost extension for the project as the length of time required to identify the appropriate antigen for our ISAV recombinant vaccine is more than we had initially anticipated.
Impacts
In the first year of the project, we conducted in vivo studies using the Phase I vaccines that we had identified as efficacious. We found that one of the recombinant vaccines, while not eliciting a measurable humoral immune response in immunized fish, nevertheless resulted in 61% relative percent survival when fish were challenged with virus 600 degree days after the first immunization and 258 degree days after a second immunization with the same vaccine. The booster regimen was critical to the vaccine efficacy as fish receiving a single dose of the same vaccine were not protected upon virus challenge. Our goal in the second and third years of the project was to increase the reliability, potency and efficacy of the vaccine. A total of 34 recombinant clones consisting of epitopes from ISAV antigens were constructed and of those, 30 antigens were successfully expressed in Escherichia coli. Large-scale expression and purification of these antigens resulted in enough
protein to formulate vaccines and efficacy studies were conducted in Atlantic salmon to assess various antigen doses, adjuvants and antigen combinations. We identified four promising ISAV antigens which were used to design vaccines consisting of the four ISAV epitopes combined with T cell epitopes and/or a molecular adjuvant. These vaccines are currently being tested for efficacy in Atlantic salmon. The successful development of an efficacious ISA virus vaccine would positively impact several areas of public interest. An ISA virus vaccine would eliminate or reduce the outbreaks of ISA in farmed salmon, increase production yields as well as reduce crop losses and feed and maintenance costs. Furthermore, the salmon aquaculture industry in Maine has created jobs in remote and rural areas of the state. These jobs, worth $140 million in personal income annually, are threatened by recurrent ISA outbreaks. Thus, an efficacious ISA virus vaccine would provide an effective disease management
tool that in combination with good fish health practices would ensure the continued growth and stability of the aquaculture industry in Maine as well as Atlantic Canada. It also seems evident that control measures, such as efficacious vaccines that reduce the viral load in farm sites, would lessen the potential for transmission of the virus between wild and farmed Atlantic salmon. In this sense, an efficacious ISA virus vaccine could form an integral component of adaptive management strategies for the protection and recovery of endangered Atlantic salmon in Maine as well as stock enhancement and continued productive commercial and recreational fisheries.
Publications
- No publications reported this period
|
Progress 10/01/04 to 09/30/05
Outputs
The ultimate goal of the Phase II project is to develop a safe, potent and efficacious ISA virus vaccine. The objectives of Phase II are to examine the effect of different vaccine doses, vaccination regimes, combinations and arrangements of antigens and immunostimulatory epitopes and adjuvant formulation on vaccine efficacy, to determine the kinetics and duration of the protective immune response and to establish the level of cross-strain protection elicited by the vaccine(s). The Phase II research plan is divided into five task areas: (1) construction of recombinant ISA virus vaccine constructs; (2) expression of recombinant ISA virus proteins; (3) purification of recombinant ISA virus proteins; (4) vaccine formulation, sterility, safety and stability; (5) vaccine efficacy studies. Three vaccine performance studies were conducted in the first year of the project. The purpose of these studies was to evaluate antigen combinations, antigen dose and the effect of
adjuvant. Two studies were also conducted to determine the susceptibility of convalescent fish to ISA virus re-exposure. Throughout these studies, the humoral immune response, virus clearance and the virus carrier status of survivors were monitored. The mid-term results indicate that the efficacy of the recombinant ISA virus vaccine(s) is dependent on vaccine adjuvant, time between vaccination and challenge and regimen of immunization. We found that one of the recombinant vaccines, while not eliciting a measurable humoral immune response in immunized fish, nevertheless resulted in greater than 60 percent relative percent survival when fish were challenged with virus 600 degree days after the first immunization and 258 degree days after a second immunization with the same vaccine. The booster regimen was critical to the vaccine efficacy as fish receiving a single dose of the same vaccine were not protected upon virus challenge. Virus was not isolated from those fish that received the
booster vaccination and survived the challenge. In addition, these fish were completely protected from a second exposure to live virus. None of the vaccines elicited a measurable antibody response in vaccinated Atlantic salmon. However, ISA virus-specific antibodies were present in sera collected from salmon 31 or 43 days after exposure to live virus and also at 114 or 145 days after exposure to virus, prior to re-exposure to virus. The highest ISA virus-specific antibody titers were observed 31 or 44 days after re-exposure of convalescent fish to live virus. The presence of ISA virus in Atlantic salmon surviving challenge was determined by cohabitation of ISA convalescent fish with naive Atlantic salmon. The convalescent fish used in the cohabitation study had not been exposed to ISA virus for at least 3 months. Tissue samples from five convalescent fish were collected at the time of cohabitation and examined for the presence of live virus by cell culture. No virus was cultured from
the tissue samples and yet 72 percent of the naive Atlantic salmon died during the course of the cohabitation study suggesting that live virus was present in the convalescent fish.
Impacts
The development of an efficacious ISA virus vaccine would positively impact several areas of public interest. An ISA virus vaccine would eliminate or reduce the outbreaks of ISA, increase production yields, decrease crop losses, savings in feed and finally maintenance savings. Furthermore, the salmon aquaculture industry in Maine has created jobs in remote and rural areas of the state. These jobs, worth $140 million in personal income annually, are threatened by recurrent ISA outbreaks. Thus, an efficacious ISA virus vaccine would provide an effective disease management tool that in combination with good fish health practices would ensure the continued growth and stability of the aquaculture industry in Maine as well as Atlantic Canada. An effective vaccination program also ensures that cultured salmon and salmon products will continue to be an accessible source of protein in both first and third world countries. This factor is especially relevant given the decline of
the natural fisheries and the projected growth of the global population to 8 billion people by 20254. It also seems evident that control measures, such as efficacious vaccines that reduce the viral load in farm sites, would lessen the potential for transmission of the virus between wild and farmed Atlantic salmon. In this sense, an efficacious ISA virus vaccine could form an integral component of adaptive management strategies for the protection and recovery of endangered Atlantic salmon in Maine as well as stock enhancement and continued productive commercial and recreational fisheries.
Publications
- No publications reported this period
|
|