Progress 07/01/18 to 02/29/20

Outputs
Target Audience: Nothing Reported Changes/Problems:A 12-month, no-cost extension of the grant was requested and granted. The new end date of the grant was Feb 28, 2020. The extension was necessary due to the seasonal nature of specimens, and small delays in finalizing study design. The extra time was used for completion of the trial as originally designed, analysis of the samples gathered from the specimens, and review of the results. During the no-cost extension period, we used remaining funds as described in the original proposal. There was no change in the project's originally approved scope of work. One aim of this project was to assess the response of the innate immune system by measuring cytokine levels in various tissues following vaccination. To do this, we designed and ordered primer-probe sets for qPCR that would detect levels of specific cytokines in head kidney, spleen, and gut tissue collected from fish before and after prime inoculation. We first tested our reagents and protocols on fish samples we had collected from a previous trial in order to verify that we could get reproducible results in both positive and negative controls. Unfortunately for the current project, the mortality event that occurred following prime inoculation precluded us from collecting the necessary samples as we could not sacrifice any more fish for this purpose. Surviving fish were necessarily reassigned to other groups in lieu of sample collection, which would have required euthanasia. Hence, we do not have any data to report for Task 3. What opportunities for training and professional development has the project provided?During the course of this project, lab aides were trained in the performance of ELISA assays and RT-qPCR. 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? Task 1: Inoculation of rainbow trout with experimental spirulina-VLP vaccines and assessment of toxicity In partnership with OSU, we obtained 795 female diploid Rainbow Trout for a trial to test a novel spirulina-based IHNV subunit vaccine. OSU inoculated fish as scheduled by the protocol, made observations on morbidity and mortality, and collected mucus and serum samples at scheduled time points for analysis by Lumen. All samples were transferred to Lumen and stored appropriately per protocol until time of analysis. Data were collected on fish mortalities over the course of the study. Following the first inoculation, we observed mortalities associated with fish vaccinated by IP injection with spirulina. Of the 20 fish included in each group (with at least 3 replicate groups for each treatment), an average of 15 fish remained for the groups treated with SP1 (control spirulina expressing "empty" or wildtype virus-like particle (VLP)), and an average of 8 fish remained for the groups treated with SP2 (spirulina expressing IHN-VLP). This translated to 25% and 60% mortality, respectively, for SP1 and SP2. Fish treated with PBS+adjuvant, either IP or PO, had less than 5% mortality. No adverse reactions were seen in fish treated orally with spirulina at any time point in the trial. Following the mid-point boost, we observed 35% mortality in the IP SP1 group. There was 0% mortality observed in all other groups, including the IP SP2 group. Due to the observed mortalities in spirulina IP injected fish following the first inoculations, we made adjustments to the protocol with regard to tissue sampling at the day 4 time point. Harvest of fish included in the IP SP1 or IP SP2 groups at this time point would have meant losing more fish in the overall study. We determined that it would be more informative to have enough fish to include in the challenge arm of the study, so we omitted this sample collection. We determined that injection of spirulina at the present dosage may have adverse outcomes, but that further studies to determine a dose response would be needed in order to draw an informed conclusion. Task 2: Assess clinical efficacy of an experimental spirulina-based vaccine for IHNV On day 57 of the vaccine trial, fish from all groups were injected with live virus or with MEM (modified eagle's medium) as placebo, and followed for 30 days. Daily observations were made regarding morbidity and mortality of fish that underwent challenge. Placebo-challenged fish had 100% survival rate over the course of the 30 days following challenge. Survival for fish in groups challenged with virus ranged from 20% to 65%, except for fish receiving the DNA vaccine (positive control), which had 100% survival rate. For other groups: Within the PO prime → PO boost treatment groups, fish receiving PBS had a higher survival (50%) than fish receiving spirulina expressing IHN-VLP (37%) or spirulina expressing empty VLP (20%). Within the IP prime → IP boost treatment groups, PBS vaccinated fish showed 27% survival, while fish receiving spirulina expressing IHN-VLP or empty VLP had survival percentages of 43% and 50%, respectively. In the IP prime → PO boost regimens: PBS primed → PBS boosted fish had 40% survival, while the PBS primed → IHN-VLP boosted fish fared only somewhat better at 63%. Fish IP primed and PO boosted with spirulina expressing empty VLP, had the highest survival of all non-DNA vaccinated fish, with 67% survival. Fish IP primed with IHN-VLP and PO boosted either with IHN-VLP or with empty VLP both had a survival rate of 43%. These data show that fish vaccinated with spirulina expressing IHN-VLP are not protected following virus challenge with IHNV. Task 3: Assess activation of the innate immune system in relevant tissues by RT-qPCR Nothing to report. Task 4: Measure the humoral immune response to vaccination by ELISA We performed ELISA analysis for all serum and mucus samples collected over the course of the study. Data include IgM recognition of IHN antigen by sera and IgT recognition of IHN antigen by mucus collected from fish during the study. Fish were organized into groups based on their prime-boost regimen. For priming, 8 separate groups were established in which individual fish were given one of the following treatments: PBS, PBS+adjuvant, spirulina expressing the empty VLP, or spirulina expressing the VLP-IHN fusion. For each of those 4 treatments, fish were inoculated either orally or by IP injection, for a total of 8 treatment groups. A 9th group was treated IP with the commercial DNA vaccine, and a 10th group of naïve fish served as negative controls. 26 days following the prime inoculation, sera and gut mucus were collected and analyzed by ELISA to determine the IgM or IgT, respectively, response to the IHN antigen, the empty VLP, or to MBP (maltose binding protein, used as ELISA negative control). All fish were negative for specific IgT to all antigens tested. For IgM, all fish were negative to the IHN antigen and to the MBP negative control. For serum IgM antibodies specific to the empty VLP, the results were as follows: PO PBS - all fish negative PO PBS + adjuvant - all fish negative PO empty VLP - 2/4 fish positive PO IHN-VLP - all fish negative IP PBS - 1/6 fish positive IP PBS + adjuvant - all fish negative IP empty VLP - 5/6 fish positive IP IHN-VLP - 6/6 fish positive On days 28, 29, or 30, fish were given a booster vaccine in accordance with the previously described homologous or heterologous prime-boost regimen. Fish that had been primed by IP injection were divided further into subgroups that were boosted either orally (heterologous regimen) or by IP injection (homologous) with PBS, spirulina containing empty VLP, or spirulina containing the VLP-IHN antigen. All fish that were orally primed, were boosted orally with the same treatment. On day 56, sera and gut mucus were collected and analyzed by ELISA for response to IgM and IgT, respectively. All fish were negative for specific IgT to all antigens tested. For IgM, all fish were negative to the IHN antigen and to the MBP negative control. For serum IgM antibodies specific to the empty VLP in the heterologous groups: IP/PO empty VLP/empty VLP - 4/6 fish were positive IP/PO IHN-VLP/empty VLP - 5/6 fish were positive IP/PO IHN-VLP/IHN-VLP - 6/6 fish were positive IP/PO PBS/PBS - 9/9 fish were negative IP/PO PBS/empty VLP - 1/9 fish was positive IP/PO PBS/IHN-VLP - 1/9 fish was positive In the homologous groups: IP/IP empty VLP - 6/6 fish were positive IP/IP IHN-VLP - 6/6 fish were positive IP/IP PBS - all fish were negative IP/IP PBS + adjuvant - all fish were negative PO/PO PBS - all fish were negative PO/PO PBS +adjuvant - 1/9 fish was positive PO/PO empty VLP - 1/4 fish was positive PO/PO IHN-VLP - all fish were negative ?ELISA results suggest that fish are able to mount a strong antibody response to antigen expressed in spirulina when injected, but not when ingested orally. Furthermore, the response we observed was directed toward the virus-like particle, at the expense of the epitopes chosen for inclusion in the VLP presentation scaffold. This may have been caused by an inadequate presentation of the insert by the VLP or by something indigenous to the fish immune system. The latter explanation is contrary to what we have observed in mammals, where immune response to the VLP is weakened in the presence of a fused antigen within its presentation platform. Fish vaccinated with the commercial DNA vaccine also did not mount an immune response to our antigen. The epitopes chosen for this study are small subsets of the protein coded for by the DNA vaccine. It is possible that the immune response to the latter does not include a robust response to the specific epitopes for this study. Task 5: Assess the specific IHN viral neutralization properties of serum and mucus Nothing to report.

Publications

Progress 07/01/18 to 06/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems:A 12-month, no-cost extension of the grant was requested and granted. The new end date of the grant isFeb 28, 2020. The extension was necessary due to the seasonal nature of specimens, and small delays in finalizing study design. The extra time is being used forcompletion of the trial as originally designed, analysis of the samples gathered from the specimens, and review of the results. During theno-cost extension period, we are usingremaining funds as described in the original proposal. There will be no change in the project's originally approved scope of work. What opportunities for training and professional development has the project provided?During the course of this project, lab aides were trained in the performance ofELISA assays. 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?We are proceeding with the plannedRT-qPCR experiments (Task 3) in order to assess activation of the innate immune system in relevant tissues.

Impacts
What was accomplished under these goals? Task 1: Inoculation of rainbow trout with experimental spirulina-VLP vaccines and assessment of toxicity In partnership with OSU, we obtained 795 female diploid Rainbow Trout for a trial to test our spirulina-based IHNV vaccine. OSU inoculated fish as scheduled by the protocol, made observations on morbidity and mortality, andcollected mucus and serum samples at scheduled time points for analysis by Lumen. All samples were transferred to Lumen and stored appropriately until time of analysis.Data were collected on fish mortalities over the course of the study. Following the first inoculation, we observed mortalities associated with fish vaccinated by IP injection with spirulina. Of the 20 fish included in each group (with at least 3 replicate groups for each treatment), an average of 15 fish remained for the groups treated with SP1 (spirulina expressing empty VLP), and an average of 8 fish remained for the groups treated with SP2 (spirulina expressing IHN-VLP). This translated to 25% and 60% mortality, respectively, for SP1 and SP2. Fish treated with PBS+adjuvant, either IP or PO, had less than 5% mortality. No adverse reactions were seen in fish treated orally with spirulina at any time point in the trial. Following the mid-point boost, we observed 35% mortality in the IP SP1 group. There was 0% mortality observed in all other groups, including the IP SP2 group. Due to the observed mortalities in spirulina IP injected fish following the first inoculations, we made adjustments to the protocol with regard to tissue sampling at the day 4 time point. Harvest of fish included in the IP SP1 or IP SP2 groups at this time point would have meant losing more fish in the overall study. We determined that it would be more informative to have enough fish to include in the challenge arm of the study, so we omitted this sample collection. We determined that injection of spirulina at the present dosage may have adverse outcomes, but that further studies to determine a dose response would be needed in order to draw an informed conclusion. Task 2: Assess clinical efficacy of an experimental spirulina-based vaccine for IHNV On day 57 of the vaccine trial, fish from all groups were injected with live virus or with MEM (modified eagle's medium)as placebo, and followed for 30 days.Daily observations were made regarding morbidity and mortality of fish that underwent challenge. Placebo-challenged fish had 100% survival rate over the course of the 30 days following challenge. Survival for fish in groups challenged with virus ranged from 20% to 65%, except forfish receiving the DNA vaccine (positive control), which had 100% survival rate. For other groups: Within the PO prime → PO boost treatment groups, fish receiving PBS had a higher survival (50%) than fish receiving spirulina expressing IHN-VLP (37%) or spirulina expressing empty VLP (20%). Within the IP prime → IP boost treatment groups, PBS vaccinated fish showed 27% survival, while fish receiving spirulina expressing IHN-VLP or empty VLP had survival percentages of 43% and 50%, respectively. In the IP prime → PO boost regimens: PBS primed → PBS boosted fish had 40% survival, while the PBS primed → IHN-VLP boosted fish fared only somewhat better at 63%. Fish IP primed and PO boosted with spirulina expressing empty VLP, had the highest survival of all non-DNA vaccinated fish, with 67% survival. Fish IP primed with IHN-VLP and PO boosted either with IHN-VLP or with empty VLP both had a survival rate of 43%. These data show that fish vaccinated with spirulina expressing IHN-VLP are not protected following virus challenge with IHNV. Task 3: Assess activation of the innate immune system in relevant tissues by RT-qPCR Nothing to report. Task 4: Measure the humoral immune response to vaccination by ELISA We performed ELISA analysis for all serum and mucus samples collected over the course of the study.DataincludeIgM recognition of IHN antigen by seraand IgT recognition of IHN antigen by mucus collected from fish during the study. Fish were organized into groups based on their prime-boost regimen. For priming, 8 separate groups were established in which individual fish were given one of the following treatments: PBS, PBS+adjuvant, spirulina expressing the empty VLP, or spirulina expressing the VLP-IHN fusion. For each of those 4 treatments, fish were inoculated either orally or by IP injection, for a total of 8 treatment groups. A 9th group was treated IP with the commercial DNA vaccine, and a 10th group of naïve fish served as negative controls. 26 days following the prime inoculation, sera and gut mucus were collected and analyzed by ELISA to determine the IgM or IgT, respectively, response to the IHN antigen, the empty VLP, or to MBP (maltose binding protein, used as ELISA negative control). All fish were negative for specific IgT to all antigens tested. For IgM, all fish were negative to the IHN antigen and to the MBP negative control. For serum IgM antibodies specific to the empty VLP, the results were as follows: PO PBS - all fish negative PO PBS + adjuvant - all fish negative PO empty VLP - 2/4 fish positive PO IHN-VLP - all fish negative IP PBS - 1/6 fish positive IP PBS + adjuvant - all fish negative IP empty VLP - 5/6 fish positive IP IHN-VLP - 6/6 fish positive On days 28, 29, or 30, fish were given a booster vaccine in accordance with the previously describedhomologous or heterologous prime-boost regimen. Fish that had been primed by IP injection were divided further into subgroups that were boosted either orally (heterologous regimen) or by IP injection (homologous) with PBS, spirulina containing empty VLP, or spirulina containing the VLP-IHN antigen. All fish that were orally primed, were boosted orally with the same treatment. On day 56, sera and gut mucus were collected and analyzed by ELISA for response to IgM and IgT, respectively. All fish were negative for specific IgT to all antigens tested. For IgM, all fish were negative to the IHN antigen and to the MBP negative control. For serum IgM antibodies specific to the empty VLP in the heterologous groups: IP/PO empty VLP/empty VLP - 4/6 fish were positive IP/PO IHN-VLP/empty VLP - 5/6 fish were positive IP/PO IHN-VLP/IHN-VLP - 6/6 fish were positive IP/PO PBS/PBS - 9/9 fish were negative IP/PO PBS/empty VLP - 1/9 fish was positive IP/PO PBS/IHN-VLP - 1/9 fish was positive In the homologous groups: IP/IP empty VLP - 6/6 fish were positive IP/IP IHN-VLP - 6/6 fish were positive IP/IP PBS - all fish were negative IP/IP PBS + adjuvant - all fish were negative PO/PO PBS - all fish were negative PO/PO PBS +adjuvant - 1/9 fish was positive PO/PO empty VLP - 1/4 fish was positive PO/PO IHN-VLP - all fish were negative ELISA results suggest that fish are able to mount a strong antibody response to antigen expressed in spirulina when injected, but not when ingested orally. Furthermore, the response we observed was directed toward the virus-like particle, at the expense of the epitopes chosen for inclusion in the VLP presentation scaffold. This may have been caused by an inadequate presentation of the insert by the VLP or by something indigenous to the fish immune system. The latter explanation is contrary to what we have observed in mammals, where immune response to the VLP is weakened in the presence of a fused antigen within its presentation platform. Fish vaccinated with the commercial DNA vaccine also did not mount an immune response to our antigen. The epitopes chosen for this study are small subsets of the protein coded for by the DNA vaccine. It is possible that the immune response to the latter does not include a robust response to the specific epitopes for this study. Task 5: Assess the specific IHN viral neutralization porperties of serum and mucus Nothing to report.

Publications