Non Technical Summary
Aquaculture, farming fish and other aquatic organisms, is expected to provide over 50% of the world's fish consumption by 2030. However, one of the biggest challenges the aquaculture industry faces is the spread of infectious diseases, which can cause significant losses in fish populations and affect the overall productivity and profitability of fish farms. One such disease, lactococcosis, caused by the bacteria Lactococcus species, poses a serious threat to trout farming in the United States. This disease leads to high mortality rates in fish, causing economic hardships for farmers and potentially impacting human health. Currently, no licensed vaccines are available in the US to protect fish from this disease, making it critical to find effective solutions to safeguard the aquaculture industry.This project addresses this urgent need by developing a novel diatom-based oral vaccine to protect fish against lactococcosis. Diatoms are microalgae naturally consumed by fish and can be genetically engineered to produce vaccine proteins. These diatoms are incorporated into fish feed, making vaccination a simple and cost-effective process. The project's approach involves expressing antigen-adjuvant fusion proteins in diatoms, which, when fed to fish, stimulate their immune system to provide protection against the disease. This method is easier to administer than traditional injection-based vaccines and reduces stress and potential side effects in fish, ensuring better health and growth performance.The ultimate goal of this project is to develop an efficient, affordable, and user-friendly vaccine that can be widely adopted by the aquaculture industry to prevent disease outbreaks. By achieving this, the project aims to enhance food security, support the economic sustainability of fish farms, and contribute to the overall health of aquatic ecosystems. The successful implementation of this vaccine can revolutionize fish health management, reduce the reliance on antibiotics, and help meet the growing global demand for sustainable and nutritious seafood. The societal benefits include increased food production, improved livelihoods for fish farmers, and a healthier environment, aligning with broader goals of agricultural resilience and community well-being.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3711	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3711 - Trout;

Field Of Science
1090 - Immunology;

Goals / Objectives
The primary goal of this project is to develop a bio-manufacturing system for diatom-based feed vaccines that will enhance US food security by integrating microalgal biomass into aquaculture feed. This innovation aims to create a cost-effective, self-adjuvanting oral aquaculture vaccine to improve resistance against endemic, emerging, and re-emerging pathogens. By commercializing this technology, PhycoVax aspires to pioneer the market for oral fish vaccines, potentially revolutionizing aquaculture health management.Comprehensive development of diatom expression methods:Achieve high expression levels of adjuvant-antigen fusion proteins in diatom cells.Develop a dual gene expression cassette controlled by two distinct promoters for a higher accumulation of recombinant proteins for enhanced immune response while minimizing incorporation into the extruded feed pellet.Fish immunization and challenge studies:Conduct oral immunization trials by feeding diatom-expressed vaccines to rainbow trout to define, refine, and validate a vaccination protocol, including feed preparation, vaccine dosages, and frequency of oral administration.Generate substantial data on the efficacy of lactococcosis prevention in rainbow trout.Pilot immunization trial at a commercial trout hatchery:Test the best-performing vaccine in a real-world setting with a history of lactococcosis outbreaks.Collect data on vaccine efficacy, immune response, and overall fish health.Establish strategic steps for commercialization:Conduct scale-up studies to ensure consistency and efficiency in manufacturing.Implement quality assurance and control measures.Evaluate product stability, efficacy, and safety.Optimize manufacturing processes and ensure regulatory compliance.

Project Methods
1. Protein Expression in Diatom:Cloning and Expression: Genes encoding the flagellin-antigen fusion protein will be codon-optimized for expression in the diatom Cyclotella cryptica. These genes will be synthesized and cloned into diatom transformation vectors using the Gateway BP cloning method. The transformation vector will include eGFP tagging for protein expression tracking.Transformation: The transformation of diatom cells will be conducted using the particle bombardment method to integrate the transgenes into the nuclear genome. Following transformation, diatom cells will be sorted using flow cytometry to select for high-level recombinant protein expression.Large-Scale Cultivation: Selected high-expressing clones will be expanded in photobioreactors. The biomass will be harvested, freeze-dried, and stored for subsequent use in fish feed formulations.2. Immunization and Evaluation:Fish Acclimation and Immunization: Rainbow trout will be acclimated and then immunized by providing an experimental diet composed of diatom biomass expressing the fusion protein. Different treatment groups will be fed varying compositions of the experimental diet to determine the optimal diatom inclusion rate and dosage for effective oral vaccination.Challenge Studies: Five weeks post-immunization, fish will be challenged by immersion in a solution containing the pathogen Lactococcus petauri. Control groups will include fish fed a commercial diet and those fed a mixture of the commercial diet and wild-type diatom cells.Monitoring and Data Collection: Fish will be monitored for clinical signs of disease, and any exhibiting severe symptoms will be euthanized and subjected to bacteriological examinations. The relative percent survival (RPS) will be calculated to evaluate vaccine efficacy.3. Data Analysis and Interpretation:Statistical Analysis: Data will be analyzed using the SAS statistical package to perform ANOVA and post hoc pairwise t-tests of least-squares means. Survival curves will be compared using a log-rank test to determine statistical significance.Immunological Assays: Enzyme-linked immunosorbent assays (ELISA) will be used to evaluate specific antibody production in the serum of immunized fish. Additional immune parameters, such as cytokine and immunoglobulin levels, will be quantified following standardized protocols.