Progress 07/01/21 to 12/31/22
Outputs
Target Audience:This project is directed at TiLV is an emerging challenge for tilapia aquaculture as a proof of concept for a platform for oral delivery to aquacultured fish and potentially crustaceans. There have only been a few outbreaks of TiLV in the USA; however, the virus has devastated other areas around the globe where tilapia culture is more extensive. Therefore, the immediate impact on the US aquaculture with an oral TiLV vaccine will only be as a prophylactic for imported broodstock. However, the development of an inexpensive oral vaccine platformapproach will have enormous value to control other pathogens of fish that are produced in the US but of lesser value. To date, vaccination of fish other than salmon have mostly been with immersion vaccines and cannot provide protection during grow out. Tilapia, catfish, carp, and trout are species grown in the US that are inneed more cost effective and simple vaccines to address viral diseases to make this industry more sustainable, humane, and economical. Success in this project could impact all these huge but underserved aquaculture sectors by providing effective and economical vaccines. The target audience for this research project are aquatic animal health practitioners as well as the extensive catfish and trout farms in the US who need better solutions to disease control at a commercially relevant price. Changes/Problems:Our initial yeast transformation system did not provide sufficient selection pressure to generate the required Ti8_6His expressing mutants. We were forced to request a NCE and shift to a different yeast expression system. This system worked well and provided verified clones expressing the Ti9_6His chimeric protein. These clones were used to generate recombinant proteins used for the vaccines as well as for the immunoassays. The mucus immunoassays based on a peptide antibody against IgT has extremely high non-specific background. This is still undergoing research to determine the causes in the hope that valuable data from the posterior intestine can be generated in the near term. Covid-19 limited our access to the laboratory and hindered the hiring of needed part-time help. 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?
Aim 1 - Expression of one TiLV gene in yeast, validation of expression and formulation in an oral delivery system Saccharomces cerevisae and Sf9 insect cells were used to express recombinant Ti8_6His protein. The insect cell culture was outside of what the grant and paid for by IR&D funds. Ti8_6His was cloned into pFastBacPlus vector and expressed in Sf9 or pYES3/CT vector and expressed in yeast. Nickel chelate columns were used to affinity purify for IP injection and immunoassays. Biomass containing Ti8_6His was freeze-dried, quantified for Ti8_6His protein, formulated into MSP particles with the oral adjuvant LactORN and then used to run the animal feeding trails at the UAZ. Aim 2 - Cloning of additional potential vaccine antigens from TiLV into a yeast expression system. TiLV proteins Ti3 and Ti5 genes were synthesized and cloned into the pYES3/CT vector. Mutant clones containing these genes were produced and validated for correct size and orientation of the genes. We have not done expression validation. Aim 3 - Tilapia animal trial with oral vaccine formulation plus injection of antigen The fish trial was carried out in the new fish aquatic testing laboratory at UAZ. Fish were grown to about 25 g in large tanks at 80?C. Five treatments: low antigen (encapsulated or unencapsulated), high antigen (encapsulated), IP injected antigen (low) and mock injection control. Both the oral vaccine and the IP injection vaccine were delivered twice; once at the beginning of the experiment and again at day 30. Sampling: Naïve fish were collected on Day 0 as a baseline control. On day 7, 5 fish from each treatment were anesthetized, bled and the serum collected; animals were revived and returned to their respective tanks. On Day 30, 5 fish from each treatment were anesthetized, bled and the serum collected; no mortalities observed. On Day 60 and 61, experiment terminated. Fish anesthetized and placed in a plastic bag containing 1 mL of phosphate buffered saline (PBS) and gently massaged for >30 seconds. The mucus in PBS was collected and flash frozen on dry ice. The fish was then bled and placed in euthanasia solution for 2 min until sampled for tissues. Aim 4 - Analysis of immune response through enzyme linked immunoassay on serum samples and quantitative RT-PCR and histology UAZ completed feeding trail at the end of August 2022. The histology samples were retained at UAZ while samples for immunoassay and qPCR were sent to NuLode. ELISA for specific anti-Ti8 IgM in serum samples. IP injection of the Ti8 recombinant protein was expected to provide the highest value, MSPTi8 treatments (high & low) would provide an intermediate, while naked Ti8 would provide the least. The negative control tanks were never exposed to Ti8 and expected to have background signal, but there was considerable noise in the data. Comparison of the different treatments only found a significant difference between the negative control and the naked antigen (treatment 4; lo-oral unencapsulated). It is unclear why the values were so low for many of the samples and why there were positives in the negative control. It is possible that systemic IgM against Ti8 was not generated by oral feeding. However, the IP injection (treatment 5) should have induced antibody production against Ti8. This was a disappointing result. Production of anti-tilapia IgT peptide antibodies - NuLode internally funded production of three polyclonal rabbit peptide antibodies to hydrophilic regions of the tilapia IgT heavy chain. It is important to understand how the mucosal immune response and systemic immune responses are cross-talking during our oral challenge study and we hope this tool will increase the knowledge we can generate in this project and future work. Expression of Immune-related genes in the spleen during different treatments Spleen real-time RT-PCR - RNA was purified from 3 spleen samples from each tank using the PureLink RNA kit. RNA was converted to cDNA using the Applied Biosystems Reverse Transcription Kit with RNase inhibitor, and cDNA was quantified using a NanoDrop. Real-time RT-PCR was performed on an Applied Biosystems StepOne Plus instrument using their standard protocol. IgM expression was significantly higher in the low antigen MSP encapsulated treatment than the high encapsulated treatment and the IP injected treatment. However, there were no other significant differences between treatments. This means that there was no significant difference between the IgM expression of low antigen treatments encapsulated or not. It is perplexing why systemic expression of anti-Ti8 IgM in the IP treatment was low when this was designed to be the positive control. Following this trend, the expression of IL1-b in the IP injection was significantly lower than both the low and high MSP, but not significantly different than the unencapsulated oral. The low and high MSP did not show a significant difference. The expression of IgT was significantly lower in high MSP treatment vs. all other treatments, but low MSP treatment was not significantly different than low unencapsulated or IP injected. There were no significant differences in TNFα expression between any of the treatments, even though the low MSP treatment looks higher the noise between samples prevented statistical validation of any difference. There was no significant difference in the expression of hepcidin.. Expression of IFNg1 in the high encapsulated treatment was significantly higher than all other treatments. All other treatments were not significantly different. Annexin 6 expression in MSP encapsulated treatments was significantly higher than IP injection and unencapsulated treatments; the MSP treatments were not significantly different from each other. The IP injection treatment and unencapsulated treatments were not significantly different from each other (but lower than the encapsulated treatments). Expression of Immune-related genes in the posterior intestine during different treatments - PI Gene expression was done to best capture the induction of immune genes related to stimulation from the orally delivered antigen. Only hepcidin expression in the IP control was significantly lower than the expression in the naked Ti8 antigen treatment. Expression of all other genes showed no significant differences between treatments. From these data we are unable to say whether vaccination with the Ti8 orally (naked or encapsulated) or IP injected were capable of inducing a significant immune gene response in tilapia. Histological examination of spleen and liver at end of experiment. Spleen & liver samples were taken from 5 fish from each tank and placed directly into formaldehyde solution. Samples were analyzed at the UAZ Aquaculture Pathology Laboratory in December 2022. In the hepatocytes of Tank 10 (IP injection treatment) the lipid reserves were lower than all other tanks analyzed for some reason (all L1 while all other treatments were L2-L3). The exocrine pancreas congestion was low from not detected to level G1 (lowest level). Hepatic congestion not detected or lowest level G1 for all samples. The melanomacrophage centers varied from not detectable to level 3 and did not seem to correlate to specific treatments. All other categories were not detectable. Aim 5 - Final report and publication of results - With the filing of this final report, we will have partially completed Aim 5. We need to complete the analysis of the mucus samples using immunoassay method before deciding on whether to proceed with publication of these data. Right now, we do not see that we have sufficiently positive results for publication. This may change as additional tissues, mucus samples, and sera are analyzed.
Publications
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Progress 07/01/21 to 02/28/22
Outputs
Target Audience:The work being done during the first half of this Phase I project dealt entirely with the NuLode R&D team and the University of Arizona PI. We have just begun to interact with the LARTA mentor for preparing our project for commercialization in the not to distant future. Otherwise, we have not done a lot of outreach until we have some harder data to support our approach. Changes/Problems:The company has had some trouble getting technical help due both to the short term nature of this Phase I and to Covid-19 restrictions for the lab. The PI is spending a considerable amount of time (within the limits of the grant allocation) to get the cloning done. We have also recruited several different individuals who have applicable expertise that can work with NuLode on a part time basis to accomplish our goals. Basically the project is a bit delayed, but with recent cloning success we are getting back on track to completion. However, we plan on asking for a no-cost extension to allow us to better coordinate with UAZ in the feed trials and final analysis of all the data. What opportunities for training and professional development has the project provided?None How have the results been disseminated to communities of interest?None What do you plan to do during the next reporting period to accomplish the goals?We are close to getting the materials needed to proceed to Aims 3-5. We are focusing on getting Aim 1 completed before we do any additional cloning of TiLV genes. We need to get the animal trials going so we can begin the critical work in evaluation of the immune benefits of our oral delivery platform. By the end of the project we will have accomplished all of the stated goals and tested the ability of the MSP delivery platform vs. purified antigen injected in stimulating the immune response (both systemic and mucosal). The overall study will be published as soon as practically possible while protecting any IP that is generated during this work.
Impacts
What was accomplished under these goals?
So far the efforts have mostly been focused on Aim 1. Progress has been slower than anticipated, but we have just recovered yeast clones that have been engineered for expresion of the TiLV segment 8 protein linked to a 6 his tag for both identification (with anti-6His antibodies) and for affinity purification of the protein for use in the animal trial (as purified control) and for use in the immunoassay to look for neutralizing antibodies produced in tilapia. We are also cloning the Ti8_6His into a site in the yeast transfer plasmid that will produce yeast that retain the TiLV segment 8 protein (Ti8) with a His tag (Ti8_6His) in the biomass for use in the feeds to be used in this project. The second set of transformations did not work (bacterial) and we are repeating the cloning to assure success. As a work around, we contracted with Genewiz (Azenta US) to make these genes synthetically and have just recieved these genes for another track to getting the yeast clones completed. The Kluveremyces lactis (yeast host) auxotrophic mutants were found to be not under selection pressure (lack of fixed nitrogen) and we have been working to determine where the contaminating nitrogen is coming from (troubleshooting). Our first try used highly purified agar and still had a problem, although the control transformation had one in ten transformants expressing the expected PCR product. We are going to change to purified water in the hopes to exclude this as a potential source of trace fixed nitrogen (we are on a well system). Additionally, we are planning on getting an alternative yeast expression system based on uracil auxotrophs (Saccharamyces cerevisiae) which we have used in the past and will not be impacted by trace nitrogen. This is a huge stumbling block as the yeast system is needed for production of the feeds. UAZ expressed Ti8-6His in insect cell culture and NuLode has purifiedthe 6His tagged Ii8 protein using Ni columns. The yields of protein were low from the 50 mL culture provided by UAZ, but was definately there using western immunoblotting to detect the his tag. Since UAZ is unable to produce larger amounts of the culture, they are going to send it to NuLode and we will either scale up in house or contract with a local group to produce several liters of culture to generate sufficent materials for use in the injection control treatment andin the immunoassays in Aims 3 and 4, respectively. For Aim 3, the UAZ team has gotten its fish testing building up and running and are ready for the animal trials as soon as the yeast can be produced in sufficient quantity, expression of the Ti8_6His validated, and the yeast incorporated into the feeds. We have purchased the feed base and the oral adjuvant needed to make the feeds and are waiting for completion of Aim 1 to rapidly execute this portion of the project. Aim 4, and outside of the grant funding, we produced three peptide antibodies to hydrophilic regions of the tilapia IgT geneusing an outside service. These have been completed and are now being processed by NuLode. These antibodies are unique tools for us as we compare the immune responses in the tilapia to our recombinant antigens; there are no fish anti-IgT antibodies available and certainly none for tilapia. It is important to understand how the mucosal immune response and systemic immune responses are cross-talking and we hope this tool will greatly increase the knowledge we can generate in this project.
Publications
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