Source: UNIV OF NEW MEXICO submitted to NRP
NEW MUCOSAL VACCINES FOR AQUACULTURE: INTRANASAL VACCINES FOR FINFISH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1005329
Grant No.
2015-67015-22973
Cumulative Award Amt.
$490,000.00
Proposal No.
2014-06259
Multistate No.
(N/A)
Project Start Date
Jan 15, 2015
Project End Date
Jan 14, 2019
Grant Year
2015
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
UNIV OF NEW MEXICO
(N/A)
ALBUQUERQUE,NM 87131
Performing Department
Biology
Non Technical Summary
Infectious diseases disminish the productivity and sustainability of the aquaculture industry. Disease prevention through vaccination has allowed the expansion of fish farming worldwide. The majority of the infections that affect farmed fish are initiated at the mucosal surfaces. One way to prevent mucosal infeciton is by using mucosal vaccines. This project investigates, for the first time, the use of novel mucosal vaccines delivered via the nose, in rainbow trout. We will advance our current knowledge on mucosal fish vaccines by exploiting the immune capabilities of the fish olfactory organ. Our specific aims are: 1. understand the innate immune responses in rainbow trout olfactory organ after nasal immunization 2. assess the effectiveness of nasal vaccines in trout, 3. determine the earliest age at which trout fingerlings can be vaccinated intranasally.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31108101090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
The long-term goal of this proposal is to develop new mucosal vaccines for aquaculture that will prevent important diseases and therefore increase the sustainability and profitability of the finfish aquaculture industry in the US. These new mucosal vaccines exploit the presence of a lymphoid tissue associated with the olfactory organ of teleost fish. We have developed a nasal vaccine model in rainbow trout (Oncorhynchus mykiss) that is as effective as the injected vaccine. The objectives of the proposal are to investigate the innate immune responses that take place in the trout olfactory organ following nasal vaccination, to evaluate and compare the nasal route as an effective route for finfish vaccination using a number of available vaccines and, finally, to determine the earliest nasal vaccination time for rainbow trout fingerlings. By using rainbow trout as our study model, we hope to expand the knowledge generated in this proposal to other important farmed species such as catfish or tilapia and find, in the future, delivery technologies that allow mass nasal vaccination of farmed fish.
Project Methods
Aim #1:- Innate immune cellular and functional assays of the rainbow trout olfactory organ: Five experimental groups will be included: mock control, I.N vaccinated, i.m vaccinated and immersion vaccinated. The head kidney (HK) and olfactory organ (n=6) from each experimental group will be sampled at 4h, 12h, 1, 4 and 7 days post-vaccination. Single cell suspensions of the HK will be obtained as explained in Zhang et al, 2010. In the case of the olfactory organ, the PD has adapted a protocol originally developed for the isolation of fish gut leukocytes (Salinas et al, 2007; Zhang et al, 2010). Using this protocol, we are able to obtain 2-3x106 olfactory organ cells per fish (200-300 g). The cell suspensions include some olfactory neurons, lymphocytes and myeloid cells. Flow cytometry analysis of the olfactory organ cell suspensions revealed that 51% of all B cells are IgT+ and 49% are IgM+, a similar proportion to that found in the gut (Zhang et al, 2010). Similar to the B cell analyses, the percentage of myeloid cells present in the nose or HK from control and vaccinated trout will be quantified by flow cytometry using the anti-rainbow trout pan myeloid antibody (detects monocytes, granulocytes and thrombocytes) available in the PD's lab (Kollner et al, 2012). Additionally, cell suspensions will be used in triplicate to measure natural cytotoxic activity using the procedures described in Salinas et al. 2005 and 2008. Phagocytic activity will be determined by flow cytometry as described in Li et al, 2006. Furthermore, we will sort in a cell sorter those cells that are phagocytic (FITC+). Sorted cells will be cytospun and stained with Giemsa stain. Since the innate immune assays require many cells, we will conduct these studies in adult rainbow trout (mean weight 100 g). The PD has an animal protocol (protocol number 11-100744-MCC) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of New Mexico.- Innate immune molecules present in rainbow trout olfactory organ: using the same experimental design as in 4.1.1.1, the olfactory organ and HK (n=6) will be collected at the same time points as above. The following innate immune genes will be measured by RT-qPCR: IL-1b, TNF-a, TGF-b, IL-8, IL-6, IL-10, IL-7R, IL-17, b-defensin 1-4, hepcidin, TLR 3, 5 and 13, Mx, IFN regulatory factors 2, 3 and 8. Additionally, due to the preliminary results that show that CCL19 is a major player in trout nasal immunity, the expression of this molecule will be also measured by RT-qPCR. With this, we hope to establish which innate immune factors are key to the establishment of robust and protective nasal immune responses and whether immersion vaccination also leads to simulation of nasal immunity.Aim #2:So far we have shown that nasal delivery is as effective as i.m injection in the case of live attenuated IHNV vaccine. In order to evaluate the potential of the nasal route in a number of aquaculture vaccines, we will conduct in vivo vaccination experiments followed by challenge with the live pathogen where we will compare the novel nasal route with previously established vaccination routes such as immersion and injection. All the vaccination trials that we propose to conduct are shown in Table 2. All challenges will be performed at days 7 and 28 after primary vaccination since they showed high protection levels in preliminary section 2.5. Specific antibody titres (serum and nasal mucus) and neutralizing antibodies (in the case of IHNV) will be measured for each group (n=10) 6 weeks post vaccination. Specific anti-Y. ruckeri antibody titres will be measured by ELISA using monoclonal and polyclonal anti-rainbow trout IgM, IgT and IgD antibodies provided by Dr. JO Sunyer (University of Pennsylvania). IHNV neutralizing antibodies will be measured by plaque assay using EPC cells as explained in LaPatra et al, 1993. All experiments will be conducted two independent times. It is possible that sensitivity to odorants and response to feed are altered following nasal vaccine delivery. We will monitor growth during our vaccination trials by measuring the total biomass of each group every week. Specific growth rate will be calculated as (lnWeight2−lnWeight1)/ ΔT where ΔT is the number of days between times 1 and 2.Aim #3:In a similar manner to our developmental histological study, newly hatched rainbow trout will be sampled daily (n=15) for 2 weeks, once every three days until 600 DD and once every five days until 1200 DD. A graduate student from New Mexico will visit Dr. LaPatra's laboratory and perform the samplings. Larvae heads will be cryopreserved in OCT and cryosections of the olfactory organ will be used for LCM dissection. Total RNA will be then isolated for each time point and qPCR will be performed to measure the expression of the following immune genes and cells markers: IgM, IgT, IgD, pIgR, MHC-II, TCRa, and b, M-CSFR, G-CSFR, IL-1b, TNF-a, TGF-b, IL-8, IL-6, IL-10, hepcidin, b-defensin 1-4, TLR 3, 5 and 13, Mx and CCL19. 10 pg of RNA can be obtained per 1 LCM cell with the picopure kit. For example, 1ug of total RNA would require 100,000 cells. Thus, for those cases, likely the earliest time points, when not enough cells can be harvested, we will use the RiboAmp RNA amplification kit, which increases the mRNA content by ~1000 fold. The expression of innate and adaptive immune genes will be used to test different time points when trout larvae are first amenable to nasal vaccination. We will use the live attenuated IHNV model. Protection will be compared to those obtained for the 5 g (1200 DD) trout trials performed in Aim #2 and shown in the preliminary.

Progress 01/15/15 to 01/14/19

Outputs
Target Audience:We have targeted the scientific community and stake holders via peer-reviewed publications and conference presentations, the general public and aquaculture industry via Twitter @DrSalinasLab, and the aquaculture industry via an article in World Aquaculture magazine (in prep). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, we have provided training to two different graduate students, Ali Sepahi and Aurora Kraus. Both students recently presented their work in the 14th ISDCI congress, Santa Fe, have written their own grant proposals with the help of the PD and developed their own professional networks. Ali Sepahi defended his PhD in June 2018 and obtained a pass with distinction. He is now a postdoctoral researcher at the University of Michigan, Ann Arbor The PD also taught at the Biology of Parasitism (BoP) module at the Marine Biological Laboratory (Woods Hole) in June 2018 along with a lab session on fish diseases. Her class was attended by 16 graduate students from all over the world. How have the results been disseminated to communities of interest?Yes, as mentioned earlier the PD and graduate students have presented their work in several conferences. The PD has given 4 invited talks including: UC Riverside March 2018, University of Rochester Medical School April 2018, Woods Hole June 2018, Boston University November 2018. The PD has also started a new project with Pacific NorthWest technologies to conduct large nasal vaccination trials in the field in the Spring of 2019 using coded wire tags. Social media (Twitter @DrSalinaslab) is used routinely to share any new findings, publications, talks, posters etc related to the project. Peer review papers and the article for World Aquaculture magazine will help us further disseminate our findings to the scientific community, stakeholders and industry. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have completed all proposed aims and, in this final period, published one more manuscript (Yu et al), submitted a second one (Sepahi et al, under review) and we are finishing writing the Aquaculture Magazine article where we will summarize all the practical aspects from our findings that are of interest to industry. I think our accomplishments are outstanding and have revealed new mechanisms by which nasal vaccines are so effective in fish. In the Yu et al paper we report the critical role of IgT secretion in the immune response against parasite infections of chronic nature. In the Sepahi et al manuscript, we have demonstrated new neuroimmune interactions in the nasal mucosa and dissected the innate immune responses that occur following nasal vaccination in trout. Combined, our findings will help the rationale design of nasal vaccines for finfish and provide the immunological basis for the high levels of protection observed.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Yu Y-Y, Kong W, Yin Y-X, Dong F, Huang Z-Y, Yin G-M, et al. (2018) Mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection. PLoS Pathog 14(11): e1007251. https://doi.org/10.1371/journal.ppat.1007251
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Sepahi A, Kraus A, Casadei E, Johnston CA, Galindo-Villegas J, Kelly C, Garcia D, Munoz P, Mulero V, Huertas M, Salinas I. Olfactory sensory neurons mediate ultra-rapid antiviral immune responses in teleosts in a TrkA-dependent manner. Cell Reports (under review).


Progress 01/15/17 to 01/14/18

Outputs
Target Audience:We have targeted the scientific community and stake holders via peer-reviewed publications and conference presentations, the general public and aquaculture industry via Twitter @DrSalinasLab, and the aquaculture industry via a current article in World Aquaculture magazine. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, we have provided training to two different graduate students, Ali Sepahi and Aurora Kraus. Both students have been able to present their work in several national and international meetings, have written their own grant proposals with the help of the PD and developed their own professional networks. The PD also taught at the Biology of Parasitism (BoP) module at the Marine Biological Laboratory (Woods Hole) in June 2017. Her class was attended by 15 graduate students from all over the world. How have the results been disseminated to communities of interest?Yes, as mentioned earlier the PD and graduate students have presented their work in several venues. The PD has given 3 talks in national and international scientific meetings. The PD also gave a talk at the NIFA Animal Health PD meeting in December 2017 in Chicago. Social media (Twitter @DrSalinaslab) is used routinely to share any new findings, publications, talks, posters etc related to the project. Peer review papers and an article for World Aquaculture magazine have helped us disseminate our findings to the scientific community, stakeholders and industry. What do you plan to do during the next reporting period to accomplish the goals?We only need to submit one more manuscript for Aim 2 and address the reviewers comments once they arrive for the publication that have been submitted but are not accepted yet.

Impacts
What was accomplished under these goals? We have completed all proposed experiments and we are currently working on submitting the last paper of this award. In the current granting period we have discovered a key feature of teleost nasal immunity: the ability of vaccines to trigger ultra fast CD8 immune responses in the olfactory epithelium. Importanty, we identified a new neuro-immune interaction by which a type of olfactory sensory neuron, the crypt neuron, is able to respond to viral antigens via the receptor TrkA. This interaction results in pro-inflammatory and CD8 immune cells responses in the olfactory epithelium within 15 mins of vaccination. At the same time, this interaction also down-regulations inflammation in the olfactory bulb of the fish. We believe this is a novel mechanism by which neurons and immune cells talk to each other and that will have high translational angles in the field of animal health and nasal vaccine development. These findings are currently under review (Sepahi et al). We have also finalized the proposed experiments in Aim 2 and completed the statistical analyses. We are currently writing a manuscript (Salinas et al, 2018) that will be submitted for publication in summer 2018.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sepahi A, Tacchi L, Casadei E, Takizawa F, LaPatra SE, Salinas I. CK12a, a CCL19-like Chemokine That Orchestrates both Nasal and Systemic Antiviral Immune Responses in Rainbow Trout. J Immunol. 2017 Dec 1;199(11):3900-3913. doi: 10.4049/jimmunol.1700757.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Salinas I, Magad�n S. Omics in fish mucosal immunity. Dev Comp Immunol. 2017 Oct;75:99-108. doi: 10.1016/j.dci.2017.02.010
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Ali Sepahi 1, Aurora Kraus 1, Christopher A Johnston 2, Jorge Galindo-Villegas 3, Cecilia Kelly 1, Diana Garc�a-Moreno 3, Pilar Mu�oz 4, Victoriano Mulero 2, Mar Huertas 5, Irene Salinas 1*. Olfactory sensory neurons mediate ultra-rapid antiviral immune responses in teleosts in a TrkA-dependent manner. Under review.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Discovery of regional immunity within the nasal mucosa in rainbow trout suggests a similar mechanism in human olfactory organ. sa in rainbow trout suggests a similar mechanism in human olfactory organ, Synthetic Immunity Symposium 2017, Santa Fe, NM
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Diversification of chemokine CCL19 genes in salmonids and their role in rainbow trout nasal immunity, North American Comparative Immunology Workshop, Raleigh, North Carolina, June 2017
  • Type: Other Status: Submitted Year Published: 2018 Citation: Recent advances in fish nasal vaccinology. World Aquaculture, submitted.


Progress 01/15/16 to 01/14/17

Outputs
Target Audience:Aquaculture industry: by presenting results at DAFINET meeting, Copenhagen, May 2016. Vaccinology community: by publishing the nasal vaccine safety manuscript in FSI and by showing the relvance of the mucosal tip of the olfactory organ as the immune target for nasal vaccine design. Mucosal immunology community: by publishing and characterizing for the first time mucosal microenvironments in the olfactory organ. General public: by communicating all of our findings via twitter, website and facebook. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I have trained a full time PhD student (Ali Sepahi) under this grant. Ali has now advanced candidacy and will be defending his PhD in Spring 2018. Ali also visited Dr. Scott LaPatra's laboratory and therefore has been exposed to an aquaculture industry environment. Moreover, another undergraduate student (Mariah Sanchez) has been part of this project during year 2. For the PI, this project has signified her consolidation as an independent investigator and her transition from Assistant to Associate Professor. Dr. Salinas recently completed her tenure review at the University of New Mexico. How have the results been disseminated to communities of interest?My students have presented their results in a number of national and international meetings including the University of New Mexico Biology Research, the Department graduate seminar (Ali Sepahi), the International Fish and Shellfish Immunology Conference (Portland, Maine, June 2016) where Ali had one oral presentation and one poster presentation. Ali will be attending the Aquaculture America congress in San Antonio, Texas in February 2017 where he has been accepted to give two talks and one poster. The PI has given multiple invited seminars including one at the Biology Department, University of New Mexico and one at Texas State University (October 2016). The PI has also communicated results to the public via social media including her personal website, twitter and facebook. What do you plan to do during the next reporting period to accomplish the goals?As mentioned above, we are going to complete Aim #1 by measuring two protaglandin synthesis related genes. InAim #2 by measuring antibody responses in serum and mucus of IHNV and ERM vaccinated fish. We will also measure B cell responses in NALT by immunofluorescence microscopy. In Aim#3 we need to complete the ontogeny of immune gene expression in the olfactory organ of trout. More importantly, we will have to work on publishing all these data in three new masnucripts and communicating them to industry and stakeholders at Aquaculture America in 2017.

Impacts
What was accomplished under these goals? During year 2, we continued to make significant progress towards to completion of the three aims proposed in this award. A detailed description of the results obtained from each aim is found below. AIM#1: During year 1, we characterized the kinetics of the nasal innate immune responses at the gene level by measuring a number of immune markers and cytokine genes. During year 2, re-evaluation of the microarray results published in 2014 showed that the prostaglandin response may be a key initial response in NALT following nasal vaccination. For that reason we have recently designed primers to measure expression of two prostaglandin genes 1 day, 4 days and 7 days after vaccination. Additionally, during year 1 we found that CCL19 was the main immune gene activated in NALT 4 days after nasal vaccination with IHNV. In an effort to understand the important of CCL19 we have performed extensive bioinformatics, phylogenetic and cloning experiments to get the full picture of CCL19 genes in salmonids. This work has revealed a very complex picture: rainbow trout and Atlantic salmon have seven different genes with the DCCL motif that defines CCL19. We have named these genes CCL10a1, a2, b1, b2, c1, c-like and c-short (Figure 1). CCL19-a1 is the gene that was printed in the oligomicroarray and that we observed to be highly upregulated after vaccination. However, this diversification of CCL19 genes in salmonid genomes has prompted new questions. For instance, how are the different CCL19 forms expressed in trout tissues? and how does each form respond to nasal vaccination? Since the 7 genes can be grouped into three main clusters: CCL19a, b and c, we designed primers that would amplify these three CCL19 groups. As shown in Figure 2, the constitutive tissue expression of each CCL19 form is unique. CCL19a expression is highest in mucosal lymphoid tissues. In the case of CCL19b, we observed expression in both systemic and mucosal lymphoid tissues. Finally, CCl19c expression was the lowest and was ubiquitous in immune and non-immune tissues including brain and muscle. These results further supported that CCL19a is specialized in mucosal immunity. Furthermore, when we measured the change in expression of CCL19a, b and c in the olfactory organ following nasal vaccination, we found that only CC19a is consistently up-regulated (all time points) and also the up-regulation was greater than in the case of CCL19b. CCL19c expression was down-regulated. Again, these results indicate that CCL19a is a CCL19 isoform specialized in mucosal immunity in trout. We also made recombinant CCL19a1 as stated in the changes proposed at the end of year 1. We have functionally characterized this protein in vivo and in vitro. Our results show no chemoattractive properties of the protein towards HK leucocytes in vitro, stimulation of CD8a, IFNg and CCR7 gene expression in HK leucocytes in vitro, stimulation of CD8, granulysin and IFNg expression in the olfactory organ and HK of rainbow trout following in vivo administration and increased number of CD8a T cells in the olfactory organ following in vivo administration. Overall, these results show that trout CCL19a plays an important role in cellular mediated anti-viral immunity in trout NALT. These results are still unpublished but will be part of a new manuscript to be submitted early in 2017. During experiments performed as part of Aim #1, we made an interesting observation. We observed clusters of immune cells at the apical tips of the olfactory lamellae but not on the sides, where the lamella is a neuroepithelium. The apical portion of the lamella is a mucosal epithelium, with abundant goblet cells and epithelial cells rather than neurons. Thus, we performed substantial studies that have fully characterized the differences between the tip and the side of the olfactory lamella. This study was recently published in The Journal of Immunology (Sepahi et al, 2016). In this study, we provide key information that can be applied to improve the design of nasal vaccines. In summary, our work shows that: 1.- Viral antigen uptake occurs equally at the tip and side microenvironments. 2.- The mucosal tip microenvironment expressed higher amount of key adhesion molecules such as CCL19, V-CAM and I-CAM. 3.- The localized expression of these adhesion molecules partitions the olfactory organ and attracts immune cells to the mucosal tip. 4.- Among the immune cells present in the mucosal tip, we found clusters of CD8+ cells. These cells had a phenotype of CD8 T cells and expressed unique immune markers compared to gut and head kidney CD8+ cells. 5.- The presence of a nasal CD8 T cell population indicates that beta2 integrin is likely an adhesion molecule that compartmentalizes mucosal lymphocytes in teleost fish. 6.- Tip-resident MHC class II+ cells are located significantly closer to the lumen of the nasal cavity than are their neuroepithelial counterparts, therefore having quicker access to invading pathogens. AIM #2: In this aim, we proposed to evaluate the nasal route for a number of available vaccines and compare it with other administration routes. We proposed to measure IgM and IgT responses in mucus and serum following vaccination. We have started analyzing the mucus and serum samples from intranasal (I.N) and intraperitoneal (i.p) IHNV vaccinated fish. Our first results are very encouraging and show increased secretion of IgT antibodies in nasal mucus 28 days after vaccination both by the I.N and i.p routes. These results are very interesting and support gene expression data showing nasal immune responses in IHNV vaccinated trout i.p at the gene level (see Tacchi 2014). We are currently measuring IgM responses as well as IgT and IgM responses in skin mucus and gut mucus. In Year 3 we will complete the entire dataset by analyzing the samples from ERM vaccinated fish (skin mucus, gut mucus, nasal mucus and serum). We plan to write a manuscript reporting all these results in summer 2017. Next, in order to improve nasal DNA IHNV protection (see report from year 1) we delivered the vaccine along with recombinant CCL19 into rainbow trout. This experiment showed that there is no increase in IHNV DNA vaccine efficacy delivered intranasally when using rCCL19 as an adjuvant. AIM #3: In this aim we proposed to determine the earliest nasal vaccination time in young rainbow trout. In year 1, we performed already the in vivo vaccination trials and the results have already been published in year 1 (Salinas et al, 2015; Developmental and Comparative Immunology). In that study we observed some mortalities in IN IHNV vaccinated trout at the youngest age tested. Thus, we wanted to see of nasal vaccines are safe to the central nervous system of fish. In order to do so, we measure the presence of the IHNV RNA in the brain of I.N or i.p vaccinated trout. Surprisingly, we found no viral antigen in any of the I.N vaccinated fish, but some fish tested positive in the i.p group. When we looked for cytokine gene expression changes in the brain of IHNV vaccinated trout, we found that despite the absence of viral antigens in the brain of I.N vaccinated fish, this tissue had decreased pro-inflammatory immune responses. In contrast, the brain of i.p vaccinated fish showed a pro-inflammatory immune response characterized by expression of IL1b and TNFa, which correlated with the presence of the virus in the brain tissue. These findings show that nasal vaccination is safe to the CNS of trout and were published in Fish and Shellfish Immunology (Larragoite et al, 2016). We also proposed to study the expression of immune genes in NALT during trout development and these studies have not commenced yet. We plan to perform this work in year 3. In addition to all the aforementioned published manuscripts, I have also published a review in Developmental and Comparative Immunology entitled: "Omics in teleost mucosal immunity".

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: An attenuated virus vaccine appears safe to the central nervous system of rainbow trout (Oncorhynchus mykiss) after intranasal delivery. Larragoite ET, Tacchi L, LaPatra SE, Salinas I. Fish Shellfish Immunol. 2016 Feb;49:351-4.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Tissue Microenvironments in the Nasal Epithelium of Rainbow Trout (Oncorhynchus mykiss) Define Two Distinct CD8?+ Cell Populations and Establish Regional Immunity. Sepahi A, Casadei E, Tacchi L, Mu�oz P, LaPatra SE, Salinas I. J Immunol. 2016 Oct 26. pii: 1600678.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Irene Salinas, Susana Magadan-Mompo. Omics in teleost mucosal immunity. Developmental and Comparative Immunology.


Progress 01/15/15 to 01/14/16

Outputs
Target Audience:Over the past 9 months, we have made substantial progress towards to completion of the three aims proposed. A detailed description of the results obtained from each aim is found below. AIM#1: We have performed most ofthe experiments proposed. Specifically, we completed the innate immune gene expression at early time points in the olfactory organ of intranasal and intramuscular vaccination groups. We also performed twoimmersion vaccination studies and compared them with the nasal and injection routes. We have found a hierarchical time-series of innate immune gene activation or down-regulation. As predicted, the main molecule that becomes activated (50 fold expression) is the chemokine CCL19 (Figure 1). After than, IL1b was the second most up-regulated gene in the I.N group. Interestingly, apart from this cytokine, genes could be grouped into two main clades based on their behavior. The pro-inflammatory immune response was controlled thanks to the onset of anti-inflammatory cytokine gene expression (Figure 2). Within the antimicrobial peptide genes, we found two different patterns of expression (Figure 2).Whereas beta defensin 3 belongs to cluster 1, or genes that were generally up-regulated, all other beta defensins (DB1, DB2 and DB4) are all down-regulated in the I.N group and belonged to cluster 2. Cluster 2 genes were all generally down-regulated at days 4 and 7 in the I.N group. Figure 1: Heat map showing the gene expression fold changes in the olfactory organ of rainbow trout vaccinated intranasally (I.N), intramuscularly (i.m) or by immersion (imm60s) compared to control fish at days 1, 4 and 7. Up-regulated genes are shown in yellow, downregulated genes are shown in blue. According to the expression patterns, a tree can be generated (left of the figure) showing categories of genes that behaved similarly. It is clear that CCL19 behaves differently to any other gene. These results are still unpublished but will be part of a new manuscript to be submitted in 2016. Figure 2: Heat map showing the gene expression fold changes in the olfactory organ of rainbow trout vaccinated intranasally (I.N), intramuscularly (i.m) or by immersion (imm60s) compared to control fish at days 1, 4 and 7. In this case, we removed the CCL19 data in order to better visualize the changes in the rest of the genes examined. Notice that apart from Il1b, two major clades of genes (cluster 1 and cluster 2) can be seen based on general up-regulation or down-regulation in their expression in the olfactory organ following vaccination. Due to the clear importance of CCL19 in the nasal mucosal environment we focused on studying this molecule following a number of IHNV vaccination regimes as well as enteric red mouth bacterin (ERM) nasal delivery. The results are shown in Figure 3 below. Interestingly, we observe that CCL19 is up-regulated in the olfactory organ to a certain degree following every vaccination regime, the strongest (80 fold) and quickest (day 1) increase observed in the nasal vaccine group. The two different immersion vaccination groups were instrumental at revealing that the longer the immersion regime the greater the stimulation of nasal immunity. In conclusion, we aimed to determine reliable surrogates of nasal immune protection and we can conclude that CCL19 is a very good indicator of nasal innate immunity and could be use in aquaculture to identify good mucosal vaccine candidates. These results are still unpublished but we plan to produce a manuscript in the next six months that combines all the aforementioned data. Figure 3: Kinetics of CCL19 gene expression following 5 different vaccine regimes in the NALT of rainbow trout compared to controls (upper panel). Comparison of the kinetics observed in IHNV vaccinated fish by I.N, 2 h immersion or i.m injection at days 1, 4 and 7. Overall, both mucosal routes (I.N and immersion) led to quicker onset of CCL19 responses. I.m delivery only resulted in a significant increase in CCL19 gene expression on day 7. The magnitude of the response was greater in the I.N group than any other group at days 1 and 4 (lower panel). With respect to innate cellular and functional immune parameters, we have performed all the proposed phagocytosis assays and we found no significant differences in control compared to I.N IHNV vaccinated fish at days 1 or 7 post-vaccination. These results indicate that phagocytosis is likely not involved in the protection elicited by IHNV nasal vaccination. The other innate immune parameters proposed are still in working progress. AIM #2: In this aim, we proposed to evaluate the nasal route for a number of available vaccines and compare it with other administration routes. We proposed to perform 6 different trials and we have already completed trials 1, 2, 3, 5 and 6. Thus, only trial 4 remains to be performed. Serum and mucus samples from these trials have been collected and we will perform ELISAs to measure IgM and IgT titers in Year 2. We have already published the results from trials 5 and 6 in the journal "Vaccine" (LaPatra, Kao, Erhardt, Salinas, Vaccine 2015). With respect to trial 1, the protection conferred by the I.N (intranasal) DNA IHNV vaccine was not as high as the IM (intramuscular) route but still conferred significant protection compared to controls. At the 7 day post-vaccination challenge, the DNA vaccinated group had a survival of 70%, whereas 100% survived in the IM group (black line in Figure 4 is hidden behind the green line). Unvaccinated controls showed a 52% survival rate. When we challenged at 28 days post-vaccination and we observed a survival of 92% in the IM DNA vaccinated group, 60.5% in the I.N DNA vaccinated group and 46% in the mock-vaccinated controls. Figure 4 shows the Kaplan-Meier survival curves obtained in trial 1 for 7 and 28 days challenge points. Figure 4: Kaplan Meier survival curves of rainbow trout vaccinated with the IHNV DNA vaccine intranasally (I.N), intramuscularly (IM) or mock vaccinated. PBS control (green line) represents fish that did not receive a challenge. Each treatment was run in duplicate tanks with 25 fish in each tank. Statistical analysis is currently ongoing. This result is in line with other vaccine delivery methods that have tested DNA vaccines in fish. DNA vaccines generally speaking only confer high protection when delivered intramuscularly. Even so, our results are actually encouraging and we believe that further optimization of the nasal DNA vaccine deserves our efforts. We plan to include one more trial using a mucosal adjuvant or recombinant CCL19 along with the DNA vaccine during the second year. Both experiments will be included in a new manuscript to be submitted to Vaccine. AIM #3: In this aim we proposed to determine the earliest nasal vaccination time in young rainbow trout. We performed already the in vivo vaccination trials and the results have already been published in a recent manuscript (Salinas et al, 2015; Developmental and Comparative Immunology). We also proposed to study the expression of immune genes in NALT during trout development and these studies have not commenced yet. We plan to perform this work in year 3. In addition to the aforementioned manuscripts, I have published a review paper on evolution of nasal immunity with the graduate student (Ali Sepahi) that works under this award. This was published recently in Sepahi A., Salinas I., Molecular Immunology, 2015. Furthermore, I have also produced another review paper on the mucosal immune system of fish (Salinas, I. Biology (Basel), 2015). Changes/Problems:I propose two changes. One is to make recombinant trout CCL19 and test if CCL19 alone is responsible for the protection observed by nasal vaccines. No changes in the budget are necessary. Second I would like to test the DNA vaccine again, this time with a mucosal adjuvant to be determined. This was already proposed in the original grant within the pitfalls and alternative approaches section of Aim #2, so it is not a major change. What opportunities for training and professional development has the project provided?I have trained a full time PhD student under this grant. He also visited Dr. Scott LaPatra's laboratory and therfore has been exposed to an aquaculture industry environment. Moreover, another undergraduate student has been part of this project during the first year. I was invited to give a plenary talk in Ithaca, NY, last summer as part of the continuing education session of the American Fisheries Society Fish Health Section. I have also been invited to Bergen, Norway, to talk about nasal vaccines to two different Aquatic Vaccine companies, Vaxxinova and MSD. How have the results been disseminated to communities of interest?Both my students have presented their results in a number of national and international meeting including the International Developmental and Comparative Immunology Congress, Murcia, Spain, July 2015 where my group had four talks in total, the AFS Fish Health Meeting in Ithaca, New York, July 2015, the West Regional IMBRE-IDEA meeting in Idaho, October 2015, and the University of New Mexico Biology Research Day. Dr. LaPatra is also presenting results from this grant in November 2015 in the DAFINET meeting in Denmark. What do you plan to do during the next reporting period to accomplish the goals?As explained in my report, we will continue to process samples we have already collected, complete the remaining experiments in Aim1 and 2 and publish at least two manuscripts.

Impacts
What was accomplished under these goals? We have identified the main innate immune molecules involved in the protection of nasal vaccines during early time points. Clearly the CCL19 chemokine is of major interest and deserves further investigation. We have also performed many in vivo vaccination trials including dual vaccinations showing the high protection that nasal vaccines confer in trout. Finally, we have determined the earliest time for nasal vaccination in trout larvae.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: LaPatra SE, Kao S, Erhardt EB, Salinas I. 2015. Evaluation of dual nasal delivery of infectious hematopoietic necrosis virus and enteric red mouth vaccines in rainbow trout (Oncorhynchus mykiss). Vaccine 33(6):771-6.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Salinas I, Erhardt EB, LaPatra S. 2015. Nasal vaccination of young rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis and enteric red mouth disease. Developmental and Comparative Immunology 53(1):105-111.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Sepahi A., Salinas I. The evolution of nasal immune systems in vertebrates. 2015. Molecular Immunology, In press.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Salinas I. The mucosal immune system of fish. Biology (Basel) 4:525-539.