Progress 09/01/17 to 08/31/21
Outputs
Target Audience:The target audiences reached by my efforts in this projectweretheresearchersfromuniversities,pharmaceutical and feed companies, fish farmers, andprofessionalsattendingFish Healthmeetings whowere interested in developingoral vaccines orfunctional feed additives to prevent viral diseases in fish. Changes/Problems:The aquaculture facility X, where the first trial was conducted, encountered some technical difficulties in collecting the samples and analyzing the data, hence, the second objective was not completed in time. In addition, due to the COVID-19 pandemic, research activities were abruptly halted due to Campus closures which restricted our work in the lab. As a result, the University had requested a no-cost-extension to complete the testing of oral IHNV vaccine product. However, these problems did not affect the outcome of this project and we were able to accomplish the proposed objectives. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our first objective was to clone and express the recombinant glycoprotein (G) gene of IHNV to develop an oral vaccine, using a baculovirus expression system.This goal was accomplished by cloning the host-protectiveIHNV-G genein a baculovirus transfer vector and generating a recombinant baculovirus that can produce recombinant IHNV-G protein in insect cells. Subsequently, we generated a second baculovirus construct in which we fused thetrout-specific complement activation gene fragment, C5a, (known to enhance immune response)with the IHNV-G gene. These recombinant baculoviruses, with and without the molecular adjuvant sequence (C5a), wereanalyzed using molecular biology methods to confirm the expression of the target IHNV proteins in insect cell cultures. The next objective was to produce large quantities of recombinant IHNV-G and IHNV-G-C5aproteins in Trichoplusia ni insect larvae (cabbage looper) system, andto prepare oral vaccines. This goal was achieved by infecting insect larvae (2 kg, wet weight) with individualbaculovirus, which yielded about 400 g of freeze-dried larvae (FDL) powder after the lyophilization step. Two FDL samples, harboring the host-protective IHNV-G proteins, were analyzed and quantified by molecular biology techniques to confirm that our target IHNV-G proteins were not degraded after freeze-drying and irradiation processes. These non-infectious samples, containing approximately 4% of recombinant IHNV-G protein (test feed A) and IHNV-G-C5a protein (test feed B) protein, were used without further purification as functional feed additives to test their efficacy as oral vaccines in rainbow trout. To administer the vaccines orally, commercial feed was formulated by mixing the feed with FDL powder of test feed A or B at 3% and then top-coating it with gelatin binder and fish oil. Our second major objective was to evaluate the efficacy of two oral recombinant vaccines in their ability to reduce mortality in rainbow trout, following intraperitoneal (IP) exposure to IHNV in a laboratory challenge model at 1-week post administration of booster vaccines (Challenge 1) and 4-weeks post administration of booster vaccine (Challenge 2). This study was performed on a contract basis at the aquaculture testing facility X. For each challenge point, a low and a high IHNV concentration were targeted. Unfortunately, the aquaculture facility encountered some technical difficulties in preparing the challenge inoculum and collecting the data, which resulted in high mortality in all treatment groups, including control, irrespective of challenge virus concentrations for both Challenge 1 and Challenge 2. Since this objective was still incomplete,we requested a no-cost-extension and conducted another trial at the aquaculture research facility Y. Oral vaccine test samples used in this study were identical to what was mentioned in the previous study, except that the fish were challenged following an immersion (IM) method at a low and a high IHNV concentration. Inadvertently, the aquaculture facility Y did not optimize the challenge inoculum to meet the requested titers of IHNV, which resulted in high mortality of 91.7% at day 8 in the control group, as opposed to 50% at day 15. However, our test feed B exhibited 50% mortality at day 8, suggesting that this oral vaccine can afford significant protection against IHNV infection. If we average the mortality on a day to day basis until day 10, the relative percent survival rate ofIHNV-G-C5a vaccine is 49.0%. In conclusion, this study was successful in demonstrating that oral IHNV vaccine administered in feed was effective in reducing the impact of IHNV infection and associated mortality in rainbow trout. Further studies are needed to determine theefficacy of this oral vaccine by optimizing the challenge dose ofIHNV.
Publications
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The target audiences reached by my efforts during this reporting period werethe researchersin universities and audience attending the Fish Health meeting and interested inpreventing the viral diseases and improve fishhealth. I spoke with two leading fish feed companies and explainedthe need for developingfunctionalfeed additives that can prevent viral diseases in aquaculturewithout the need of injecting fish. Changes/Problems:The aquaculture facility, where the trial was conducted, encountered some technical difficulties in collecting the samples and analyzing the data, hence,objective 2was notcompleted in time. In addition, dueto the COVID-19 PANDEMIC crisis, research activities were abruptly halted due to Campus closures which resulted in the closing of the PI's lab. Therefore,the University has requested a no-cost-extensionto complete the testing of recombinant IHNV vaccine product administered orally to rainbow trout.ThePIwill be preparing fresh vaccine samples and repeat this study, which will be completed next year.The redistribution of funds from travel to supplies will support these activities. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, (objective 2repeated), wewillevaluate the efficacy of baculovirus-expressed IHNV proteins delivered orally in reducing the impact ofIHNV infection in rainbow trout.Fish trial will be conducted with twodifferent types of IHNV antigens which will be formulated to make oral vaccinesby top-coating (3%)the feedwithfishoiland delivered orally as functional feed additives.
Impacts
What was accomplished under these goals?
Last year, we describedsusccessfulproduction of IHNV-specific proteins in insect larvae, and its conversion to freeze-dried larvae (FDL) powder. Twosamples, harboring the host-protective IHNV-G proteins, were analyzed by Western blotting to confirm thatour target IHNV-G proteins were not degraded after freeze-drying and irradiation processes. This year, we tackled the secondmajor objective of evaluating theefficacy of twooral recombinant vaccines in their ability to reduce mortality in rainbow trout, following intraperitoneal (IP) exposure to IHNV in a laboratory challenge model at 1-week post administration of booster diets (Challenge 1) and 4-weeks post administration of booster diets (Challenge 2).This study was performed on a contractbasisatthe Center for Aquaculture Biotechnologies, San Diego, CA. For each challenge point, a low and a high IHNV concentration were targeted. Unfortunately,theaquaculture facility encountered some technical difficultiesin preparing the challengeinoculum and collecting the data, whichresulted in highmortality in all treatment groups, including control, irrespective ofchallenge virusconcentrations for both Challenge 1 and Challenge 2. Since this Objective 2 was not met, we will be preparing fresh vaccine samples and repeat this studyto complete this objective in the coming fiscal year.
Publications
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:The target audiences reached by my efforts during this reporting period werethe researchersin universities and companiesworkingon functional feed additives to prevent diseases and improve animal health. I spoke with investors and venture capitalists and explained the need for the development of novel feed additives that can prevent viral diseases in aquaculture without injecting the animals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period (objective 2 continued), we willevaluate the efficacy of baculovirus-expressed IHNV proteins delivered orally in protection against IHNV infection. Fish trial will be conducted with 3 different types of IHNV antigens that will be formulated to make oral vaccines by top-coating (3%) on the feed using castor oil or microencapsulated with lipid based matrix, and delivered orally as functional feed additives.
Impacts
What was accomplished under these goals?
One of the major objectiveswas to producethe recombinant protein(s)ofIHNV in insect larvae system to develop functional feed additive (oral vaccine). This goal was completed. We generated two recombinant baculovirus harboring the host-protective IHNV-G protein and the other containing a known molecular adjuvant sequence of trout, known to stimulate immune response. Another recombinant baculovirus construct was made which expressed the IHNV-G protein that was more soluble than other two previous constructs. Insect larvae, weighing about 800 g, were infected with new baculovirus,which yielded about 150 g of freeze-dried larvae powder.We are quantifying the proteins from these constructs and working to formulate this insect powdersto make functional feed additives, whichwill be tested for its efficacy as oral vaccinein rainbow trout (objective 2 to be continued next year).
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:The target audiences reached by my efforts during this reporting period werethe faculty members andresearchersin universities, and companiesworkingon aquatic animal health diseases.I also metfish farmers in the U.S.Trout Farmers Association Conference in Twin Falls, ID,September 2017(https://ustfa.org/ustfa-2017-fall-conference/), where I gave an invitedtalk.IattendedtheEastern Fish Health Workshop in Chattanooga, TN, April 2018, and International Symposium of Aquatic Animal Health in Charlotteville, Canada, September 2018, where I talked aboutthedevelopment of oral vaccines for aqauculturewith the stakeholders and researchers at theseconferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period (objective 2),we willevaluate the efficacy of baculovirus-expressed IHNV proteins delivered orally in protection against IHNV infection. Fish trial will be conducted with freeze-dried larvae (FDL)-expressed IHNV antigens (with or without C5a adjuvant protein), which will be formulated to make oral vaccines by top-coating (3%) on the feed using castor oil. Furthermore, FDL-expressed IHNV antigenswill be encapsulated using oil-based matrix, mixed with the feed and delivered orally as vaccines.
Impacts
What was accomplished under these goals?
One of the major objectiveswas to clone and producethe recombinant protein(s)ofIHNV to develop oral vaccine, using a baculovirus expression system.This goal was accomplished. First, weclonedthe host-protectiveIHNV-G genein a baculovirus vector and generated a recombinant baculovirus. Second,we fused thetrout-specific complement activation gene fragment, C5a, (known to enhance immune response) with the IHNV-G gene, and generated anotherrecombinant baculovirus.These recombinant baculoviruses, with and without the molecular adjuvant sequence, C5a, wereused to scale-up the production of recombinant IHNV-G and IHNV-G-C5aproteins in Trichoplusia ni insect larvae (cabbage looper). Two kg ofinsect larvae (wet weight) were infected with individualbaculovirus,which yielded about 450 g of freeze-dried larvae powder.We are working to formulate this insect larvae powdersto makevaccines, whichwill be tested for its efficacy as oral vaccine(objective 2 to be conducted next year).
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