Source: AUBURN UNIVERSITY submitted to
RAPID VALIDATION OF IMMUNOGENIC TARGETS FROM HYPERVIRULENT AEROMONAS HYDROPHILA FOR DEVELOPMENT OF A RECOMBINANT PROTEIN VACCINE AGAINST VMAS IN CHANNEL CATFISH (ICTALURUS PUNCTATUS)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029275
Grant No.
2022-70007-38285
Cumulative Award Amt.
$298,403.00
Proposal No.
2022-06026
Multistate No.
(N/A)
Project Start Date
Sep 10, 2022
Project End Date
Sep 9, 2025
Grant Year
2022
Program Code
[AQUA]- Aquaculture Research
Project Director
Barger, P.
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
(N/A)
Non Technical Summary
Hypervirulent Aeromonas hydrophila (vAh) is a significant pathogen in freshwater aquaculture.Since 2009,epidemic outbreaks of motile aeromonas septicemia caused by vAhhave resulted in devastating losses in Southeastern aquaculture production facilities, and these losses have had a particularly deleterious impact in the Alabama catfish aquaculture sector which is vital to the economy of the State's most economically depressed counties.This research will advance vaccine development targeting vAh and vAh-related motile aeromonas septicemia (vMAS). Vaccination is a keystone of disease prevention in aquatic production systems and could prove an effective preventive method against vMAS. However, vaccine development is a slow process, largely due to the time required to identify and validate a suitably effective antigen that will stimulate a strong protective immune response in vaccinated catfish. This research will significantly speed up this process by pre-screening targeted bacterial proteins identified by multi-omics (protein and gene expression) research approaches. This projectwill identify promising recombinant protein vaccine candidates that stimulate robust humoral adaptive immunity in catfish by using preliminarydata to select proteins with high potential as vaccine candidates, and to develop a latex-microsphere-based agglutination assay platform to rapidly screen these immunogenic targets for antigenicity.New preliminary data generated through our researchhas identified proteins that are highly expressed and/or secreted by vAh, and has identified multiple high-priority vaccine candidate proteins. Using recombinant protein technologies and methods, multiple surface-expressed recombinant vAh proteins will be screened for the ability to induce catfish IgM antibody response through latex agglutination assays using serum from catfish previously infected with vAh. Highly immunogenic recombinant proteins identified during this research will be used in the development of subunit and multivalent subunit vaccines.An effective vaccine will offer an affordable, durable protection option to producers and significantly decrease the staggering losses resulting from vMAS each year.
Animal Health Component
45%
Research Effort Categories
Basic
10%
Applied
45%
Developmental
45%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113710104050%
3113710109050%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1040 - Molecular biology; 1090 - Immunology;
Goals / Objectives
With the overarching goal of providing producers with affordable, effective pathogen control strategies, this project willserve as the basis for development of protective vaccine(s) against hypervirulentAeromonas hydrophila(vAh) and vAh-associated motile aeromonassepticemia (vMAS)in catfish. This projectwill endeavor to identify promising recombinant protein vaccine candidates that stimulate robust humoral adaptive immunity in catfish by using preliminary omics data to select proteins with high potential as vaccine candidates, and to develop a latex-microsphere-based agglutination assay platform to rapidly screen these immunogenic targets for antigenicity.The principal aim of the proposed research is to identify secreted proteins that stimulate a strong immunogenic response in channel catfish, with the ultimate goal of developing an effective, long-lasting vaccine against vMAS. To this end, the following objectives are proposed:Objective 1Generate recombinant vAh proteins from surface-expressed proteins identified as high priority vaccine candidates based on availableomics dataObjective 2Evaluate and validate immunogenic recombinant protein targets via latex agglutination serum assays.We expect to identify multiple recombinant proteins that stimulate a strong immune response in channel catfish that can then be utilized in vaccine trials.
Project Methods
An array of secreted or surface-expressed vAh proteins will be screened for the ability to induce catfish IgM antibody response through latex agglutination assays using serum from catfish previously infected with vAh. Previous research in the PI's lab has identified vAh proteins that are highly expressed and/or actively secreted by vAh. Using recombinant protein technologies and methods, immunogenicity of these proteins, including a fimbrillin protein,two ABC transporter proteins, two outer membrane receptors,and a chitin binding protein will be assayed.Identification of target proteins:Proteins were selected based on their presence in the secretome of planktonic or biofilm-cultured samples [15] and/or upregulation of corresponding genesin vivoandex vivo[28, preliminary data]. All proteins selected for immunogenic validation are either secreted or surface-expressed proteins.Antiserum production:To generate serum antibodies to vAh ML09-119, 20 specific-pathogen-free channel catfish averaging 175 grams, reared under IACUC maintenance protocol #2018-3251, will be transferred to five challenge aquaria (four fish per aquarium) and allowed to acclimate for 1 week. Water temperature of 30°C will be maintained by in-tank heaters and airstones present in each tank will provide aeration. Fish will be fed daily and biological filtration provided by in-tank sponge filters pre-seeded with nitrifying bacteria. Following acclimation, fish will be challenged with a sub-lethal concentration (104CFU) of vAh ML09-119 by intraperitoneal injection. Fish will be monitored for signs of disease and moribund fish will be euthanized by pithing. Twenty-one days post-challenge, fish will be sedated in tricaine methanesulfonate buffered to neutrality, and blood will be collected from caudal vein, allowed to clot at 4°C overnight, and serum separated via centrifugation. Serum will stored at -20°C until use. Antibody titers will be determined by ELISA as previously described.Negative control serum will be collected from unchallenged SPF catfish.Cloning:Escherichia coliNovaBlue (Novagen) will serve as the cloning strain andE. coliBL21 (Invitrogen) will be used for expression. Plasmid pET-28a-c+ with C-terminal His-tag (Sigma-Aldrich) will serve as the expression vector.Coding region of selected proteins will be amplified from vAh genomic DNA by PCR using gene-specific primers with incorporated 5' restriction sites (Eurofins Genomics) based on vAh ML09-119 genome sequence. Each protein will be confirmed by sequencing (Eurofins Genomics). Validated sequences will be cloned into pET-28a-c+ expression vector and positive clones transformed intoE. coliBL21 for expression.Recombinant protein expression:Following transformation,E. coliBL21 containing recombinant plasmids will be cultured in Luria Broth (LB) supplemented with kanamycin in an orbital incubator at 37 °C until cultures reach an optical density (600nm) of 0.5. Recombinant protein expression will then be induced by the addition of 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture, followed by 8 h incubation at 37 °C. Cells will be pelleted by centrifugation and whole bacterial proteins will be isolated using B-PER Complete bacterial protein extraction reagent (Thermo Scientific) following manufacturer's protocol, then solubilized in Tris sample buffer for 5 min at 80 °C. Proteins will be loaded onto SDS-PAGE gels and electrophoresed to verify recombinant protein expression. Non-recombinantE. coliBL21 and an uninduced recombinant clone will serve as controls. Verified positive clones will be subcultured and cryogenically stored at −80 °C in glycerol freeze media.To purify recombinant proteins, positive clones will be cultured in Luria Broth (LB) and induced, as above. Cells will be pelleted by centrifugation and cells lysed using B-PER Complete bacterial protein reagent, as above. Soluble fractions will be removed by centrifugation. Recombinant proteins in inclusion bodies will be recovered using inclusion body isolation maxi kit (NorGen Biotek) following manufacturer's protocol. The recombinant protein suspension will be loaded onto HisPur Ni-NTA spin columns (Thermo Scientific) and eluted following manufacturer's protocol. Protein concentration will be determined spectrophotometrically at 280 nm (Synergy HTX Multi-mode reader, BioTek). Recombinant proteins will be analyzed by native SDS-PAGE. Proteins will be excised from PAGE gel and confirmed by MALDI-TOF mass spectrometry.Development of recombinant latex agglutination test:Recombinant microspheres:Polystyrene latex microspheres (0.75 micron, Thermo Scientific) will be diluted in sterile phosphate buffered saline (PBS), pH 7.2 and incubated 45 minutes.Microsphere coating: Microspheres be centrifuged, the supernatant decanted, and the particles resuspended to in a solution of recombinant protein in PBS. Optimal protein coating concentration will be determined empirically. Microspheres will be incubated with constant shaking at room temperature for 24 hours.Following coating step incubation, microspheres will be recovered by centrifugation and supernatant decanted.Microsphere blocking: Coated microspheres will be resuspended in PBS + 0.5mg/ml goat serum and allowed to block for 1 hour at room temperature with constant shaking.Blocked microspheres will be centrifuged, washed twice with PBS, and recovered recombinant protein-coated microsphere diluted to 1% with PBS containing 0.1% sodium azide, 0.05mg/ml normal goat serum, and 0.5% glycerol, and stored at 4°C until use.Agglutination assay:Agglutination reactions will be performed on black agglutination cards (De Beer Medicals) with recombinant protein-coated microspheres and fish sera at room temperature.Recombinant protein coated microspheres will be added to designated circles on agglutination cards. Serum from challenged or non-challenged (negative control) fish will be added to the coated microspheres and monitored for agglutination.Optimal assay parameters, including microsphere and serum concentrations and incubation times, will be determined empirically based on naked-eye observation of agglutination.Project Evaluation:We expect to identify multiple recombinant proteins that stimulate a strong immune response in channel catfish that can then be utilized in vaccine trials. As protein solubility is of paramount importance for biological activity, ensuring the expression of functional recombinant proteins will be an initial measure of success. As nocard-based agglutination assay is currently available for the rapid screening of vAh immunogenic targets, development and validation of this assay will be another significant measure of project success. Successful screening and identification of highly immunogenic recombinant proteins that can be used as asubunit or multi-subunit vaccinewill be the ultimate measure of project success.Project Efforts: A significant effort will include mentoring of graduate and undergraduate students inlaboratory and wet-lab instruction, and in the development and strengthening of non-technical skills. Graduate and undergraduate students will be encouraged topresent their findings to peers in scientific platforms and directly to end-users through conferences, seminars, or workshops to enhance project outreach. Endresults of theresearch will be presented at scientific conferences and submitted for publication in peer-reviewed journals. In order to make the results of this data available and useful for catfish producers and other primary stakeholders, results will be made available in non-technical summaries through Alabama Extension, Alabama Fish Farming Center, and presented at conferences commonly attended by catfish farmers, such as the Catfish Farmers of America Annual Convention.

Progress 09/10/23 to 09/09/24

Outputs
Target Audience:In the past year, this research has reached a multidisciplinary audience, ranging from basic scientists interested in the molecular mechanisms of bacterial virulence to applied veterinary professionals seeking innovative solutions to real-world aquaculture challenges. Prelimianry data waspresented at both national and local conferences of the American Society for Microbiology (ASM) and at theAuburn University College of Veterinary Medicine Research Seminar Series.At the ASM conferences, the target audience reached weremicrobiologists, molecular biologists, and infectious disease specialists who are interested in bacterial pathogenesis, antimicrobial resistance, and innovative approaches to disease management. The AUCVM Research Seminar Seriesaudienceincluded fish disease researchers, veterinary researchers, clinicians, and students who focus on aquatic animal health, zoonotic diseases, and veterinary public health, This group is particularly invested in practical applications of research findings that can directly benefit animal health and aquaculture practices. Changes/Problems:No major changes or problems have occured. What opportunities for training and professional development has the project provided?Over the past year, this project has provided an array of opportunities for training and professional development across various educational levels, including undergraduate students, graduate students, and postdoctoral researchers. This training has fostered the development of technical skills, critical thinking, and professional competencies that are vital for careers in aquaculture. Undergraduate students supported by thisfundinghave engaged in hands-on research experiences that complement their classroom learning. They have learned catfish husbandry and handling techiniques, water qualty monitoring and biofilter maintenance, and the importance of maintaining detailed records onall aspects of fish health. In the laboratory, they have learned proper research techniques, data management, troubleshooting, and how to "think like a sceintist". This exposure enhancestheir understanding of scientific methodsand provides a unique opportunity to apply critical thought to problem solving.. Additionally, undergraduates gain experience in scientifc communication by presenting updates on their work at weekly lab meeting. The postdoctoral researcher has played a pivotal role in leading many of the research activities within the projectwhile also learning advanced laboratory techniques in cloning and recombinat protein expression and purification. He has gained valuable experience in project management, mentoring of undergraduates, and interdisciplinary collaboration.He is currenlty involved with manuscript preparation, and has presented his reseearch at local and national conferences, to diverse audiences. How have the results been disseminated to communities of interest?Research has been disseminated to scientists, students, veterinarians, and aquaculture researchers through platform and poster presentations at several local and national conferences and seminars over the past year. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting persiod, work will be completed on cloning and recombinant protein expression of vaccine candidate proteins. The latex agglutination assay will continue to be validated and a standard operating protocol will be generated to ensure reproducibility of the assay across different research facilities. When the SOPs have been established, all purified recombinat proteins will be asssayed via agglutination assay to determine their propensisty to stimulate an immune response in channel catfish. Future research supported by these finding will include aquarium-based vaccination trial to determine assay antigenticty and prtoective immunity correlates.

Impacts
What was accomplished under these goals? During this reporting period, significant progress has been made toward both objectives.Mulitple high-priority vaccine candidates have been successfully cloned, expressed, andpurified. Development has begun on the latex agglutination assay using whole-cell vAh as the antigen to coat the latex beads,vAh antiserum generated in channel catfish as a positive control, and serum from specific pathogen free catfish as a negatve control. Using this method, we were able to visualize significant agglutination in the positivecontrol sample, while no agglutination was visible in the negative control sample. This supports our hypothesis thatimmunogenicity of a vAh proteinscan be determinedin vitro, and that a card-based agglutinatin assay may be a rapid, inexpensive first line assay to screen potential vaccines. We are continuing work to validate the assay using recombinant proteins, and developing a standard protocol to ensure reproducibility.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Xu T, Barger, P., Rasmussen-Ivey, C.R., Moen, F.S., Fernandez Bravo, A., Lmay, B., Beaz-Hidalgo, R., Khan, C.D., Escarpulli, G.C., Yasin, I.,S., Figueras, M.J., Sayuti, M.S., Karim, M.M., Alam, M., Thao, L.T.T, Phuong, N.H., Addo, S., Duodu, S., Ali, S., Latif, T., Mey, S., Somony, T., and Liles, M.R. 2024. The global distribution of hypervirulent Aeromonas hydrophila and its control by recombinant vaccine. In: American Society for Microbiology MICROBE; Atlanta, Georgia
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Xu T, Barger, P., Rasmussen-Ivey, C.R., Moen, F.S., Fernandez Bravo, A., Lmay, B., Beaz-Hidalgo, R., Khan, C.D., Escarpulli, G.C., Yasin, I.,S., Figueras, M.J., Sayuti, M.S., Karim, M.M., Alam, M., Thao, L.T.T, Phuong, N.H., Addo, S., Duodu, S., Ali, S., Latif, T., Mey, S., Somony, T., and Liles, M.R. 2023. The global distribution of hypervirulent Aeromonas hydrophila and its control by recombinant vaccine. In: Southeastern Branch of the American Society for Microbiology Regional Meeting; Auburn, Alabama


Progress 09/10/22 to 09/09/23

Outputs
Target Audience:Research scientistsand students Changes/Problems:While there have been no major changes in the protocol, our research hasbeen consistently plagued with supply production delays. Inability to acquire specific expression vectors has required adaptation of protocols to utilize available vectors, andpurification columns and nickel-chelating resins have been on backorder for several months. If resins are still unavailable after recombinant protein scale-up is complete, the protocol will be adapted for use with an alternate purification system. What opportunities for training and professional development has the project provided?Over the last year, three undergraduates and a post-doctoral fellow have been involved in multiple aspects of the project. One pre-veterninary medicine student received fish care and handling training andtraining in basic laboratory techniques and lab safety, Two other undergradaute studentswere also trained in fish care and handling. These students have received in-depth training in molecluar biology and proteomics techniques and have been intimatley involved in each step of the project, from in silicoanalysesto help select viable protein targets tocloning and protein expression. One of these students completed her baccalaureatedegree in May, and continued her research on this projectas a pre-graduate research assistantprior to entering anInfectious Diseases and Vaccinology doctoral program in August. This project has also offered training for a postdoctotal research fellow. The postdoc has a strong background in Fisheries, but has learned cloninig, protein expression, Western blotting, and other proteomic techniques through his involvement in this project. Furthermore, the postdoc has gained mentoring skills through his training and interactions with the undergradautes. How have the results been disseminated to communities of interest?Currently, information has only been disseminated to other scientists through informal discussions. What do you plan to do during the next reporting period to accomplish the goals?Beginning this Fall, the addition of a new PhD student tothe project will further increase our research output and will help to achieve our data dissemination goals. We will continue working on expressing individual proteins and are currently working to scale upexpression of the recombinant proteins that were successfully expressed in the pilot studies. We will then purify the recombinant proteins using His-Pur Ni-NTA protein purification columns. Purified recombinant proteins (antigen) will be used to coat latex beads at different concentrations (appropriate concentrations of antigens will be determined empirically) and antigen-coated beads will be mixed with vAh antiserum (generated in channel catfish) on latex agglutination cards to measure the degree of antigenicity of each protein. The prelimiary results of this research will be dissimenated to the scientificcommunity at the 2023 Southeastern Branch of the American Society for Microbiology (SEB-ASM) conference in November 2023, at the National ASM conference (ASM Microbe) in June, 2024, and to both researchers and producers at the 2024 Aquaculture America and Catfish Farmers of America annual conventionin February, 2024. Results will also be dissiminated through local meetingsincollaboration with the Alabama Fish Farming Center.

Impacts
What was accomplished under these goals? We are currently focusing on expressing 12 individual recombinant vAh proteinsfrom surface-expressed or secreted proteins identified as high priority vaccine candidates based on available in vivo gene expression and protein secretion data from Aeromonas hydrophila, We initiallyidentified potential protein targets from significantly upregulated genes. These protein sequencceswere screenedin silico to determine probablecellular localization, degree of hydrophobicity, and antigenic potential using EMBOSS, Protein-sol, and PSORTb analysis programs. After these analyses, 12protein targets were selected for further investigation. Primers for each target gene were designed anda touchdown PCR protocol was designed to ensure specific amplification of target genes. All genes have been successfully amplified and seqeunced. We are currently working on cloning these genes into either pET SUMO or TOPO Directional epression vectors, depending ontheir predicted solubility values. To date, six of the target genes have been clonedand sequenced to ensure the gene is in frame with the expression tag.Successfulpilot expression of threeof the 12 targetproteins has been accomplished.We have also made progress in antiserum generation, havingperformed initial sub-lethal challenges in channelcatfish.

Publications