Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The scientific community with special interests in enteric bacterial pathogens of farm animals, concerns over transmission of enteric pathogens, and especially those that are antibiotic resistant, through the food chain to human consumers, and means to prevent such transmission through the food chain by development and use of vaccines to prevent infection and colonization of such enteric pathogens in farm animals destined to produce products and/or directly to be marketed. Ultimately, the targets are vet biologics companies that would further develop and ultimately market such vaccines. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?As part of this project, seven undergraduate students from the University of Florida--Vanessa Campodonico, Alexa Sauvagere, Lex Bleckley, Diya Rana, Garima Rathor, Kavi R. Miryala, and Shivam Datta--have received training. Vanessa Campodonico and Garima Rathor were co-authors on papers published in Vaccines and Applied Sciences. Vanessa Campodonico has since been admitted to the Graduate Certificate program in Molecular and Biomedical Sciences at the FIU Herbert Wertheim College of Medicine. Alexa Sauvagere is currently interviewing for medical school. Kavi R. Miryala and Shivam Datta continue to work in the lab. How have the results been disseminated to communities of interest?Banikalyan Swain attended and presented an abstract entitled 'DESIGNING AND CONSTRUCTING OF DNA VACCINE-VECTOR SYSTEM TO PROTECT FISH AGAINST MULTIPLE INFECTIOUS DISEASES" atAQUACULTURE AMERICA 2024,February 18 - 21, 2024 San Antonio, Texas. in theAquatic Animal Health section. Banikalyan Swain gave a presentation in Phi-Zeta conference at the University of Florida,College of Veterinary Medicine. Title of the abstract:Self-destructing Edwardsiella vaccine vector system to prevent Aeromonas hydrophila and Edwardsiella piscicida infection in catfish. What do you plan to do during the next reporting period to accomplish the goals?We have vaccinated tilapia with the RAEV-TiLV DNA vaccine strains χ16048 (pG8R9050), χ16048 (pG8R9051), χ16048 (pG8R9052), χ16048 (pG8R9055), and the control strain χ16048 (pYA4545) using both intracoelomic (i.c.) injection and bath immersion methods. The vaccinated fish were subsequently challenged with virulent TiLV. This experiment is currently ongoing. Once completed, we will perform data analysis to evaluate the protective immunity conferred by the newly constructed RAEV-TiLV DNA vaccine strains. We are collecting tissue and serum samples from both vaccinated and control fish. The neutralizing antibody titers in the serum against TiLV will be determined using a neutralization assay. The titer will be calculated as the 50% endpoint of serum dilution that inhibits the cytopathic effect (CPE) in inoculated cells. Additionally, we will analyze the expression of immune-related genes critical to immune protection. Specifically, we will investigate the responses of genes encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) in immune organs such as the gills, kidney, intestine, spleen, and blood using qRT-PCR, following our established protocols. The activation of proinflammatory cytokines plays a pivotal role in regulating immunoglobulin synthesis in teleosts. Understanding the gene expression profiles following vaccination is therefore essential. Once these studies are completed, we will have achieved all the proposed objectives. Following data analysis, we plan to publish our findings in reputed open-access journals.

Impacts
What was accomplished under these goals? (1) Design of DNA vaccine vectors encoding TiLV gene segment. Sequence analysis: The TiLV genome consists of ten genomic segments encoding ten distinct proteins. Each protein has been characterized in detail with respect to its structural features (e.g., α-helices, β-sheets, turns, and coils), hydrophobicity, surface accessibility, and potential antigenicity using B- and T-cell epitope prediction tools. Each gene sequence was modified to include a C-terminal His-tag, facilitating monitoring of fusion protein synthesis via a monoclonal antibody specific to the His-tag. TiLV virus cultivation, RNA isolation and cDNA preparation and cloning of TiLV-ORFs: The TiLV isolate (WVL18053-01A) was used for this study and has been described previously (Al-Hussinee et al. 2018). TiLV isolate from a frozen stock inoculated onto a 175 cm2 flask containing confluent striped snakehead (SSN-1; E11 clone) cells. The SSN-1 cells were maintained at 25? and grown in L-15 media (Leibovitz; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) with 1× antibiotic/antimycotic (AA; Gibco, USA), resulting in a concentration of 100 IP penicillin ml-1, 100 µg streptomycin ml-1, and 0.25 µg amphotericin B ml-1. After CPE was complete, the supernatant was clarified by centrifugation at 5000 × g (20 min at 10°C). Total RNA was isolated from the virus infected cell supernatant was by using TRIzol reagent (Invitrogen, USA), and reverse transcribed into first-strand cDNA using RevertAid First Strand cDNA Synthesis kit (Thermo Scientific). According to the full-length sequence of TiLV segments (GenBank under accession no. MH319378 to MH319387) the specific primers were designed for PCR amplification. Using prepared cDNA as template, all the segments of TiLV were successfully amplified. The PCR product was purified and ligated into CloneJET vector by using CloneJET PCR Cloning Kit (Thermo Scientific). Selected clones were confirmed by PCR and sequencing. Construction of pcDNA3.1/A-TiLV DNA vaccine vectors encoding TiLV gene segment: The TiLV gene segments were initially cloned into the pcDNA3.1/A vector. The resultant plasmids were numbered from pG8R9029 to pG8R9037 (Table 1). HEK293T cells were transfected with these pcDNA3.1/A-TiLV constructs using Lipofectamine 2000 and incubated at 37 °C with 5% CO?. At 24 and 48 hours post-transfection, cells were harvested and lysed in sample loading buffer. The lysates were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a nitrocellulose membrane for Western blot analysis. The His-tagged TiLV antigens were detected using an anti-His-tag antibody, with SuperSignal™ West Pico PLUS Chemiluminescent Substrate employed to visualize the blot. Positive signals were observed for cells transfected with pG8R9029, pG8R9030, pG8R9031, and pG8R9035, confirming successful antigen synthesis. Table-1 Construction of pYA4545-Kozak-TiLV vector: The TiLV segments 2 to 9, including a C-terminal His-tag sequence, was cloned into the pYA4545 DNA vaccine plasmid between the KpnI and XhoI restriction sites. The plasmids were numbered from pG8R9050 to pG8R9057 (Table 2). A Kozak sequence (GCCACC) was introduced into each construct upstream of the start codon (ATG) to enhance translation efficiency, and the +4 nucleotide was modified to G for optimal expression. The plasmids contain several functional elements, including the rrfG, trpA, and 5S ribosomal RNA transcriptional terminators; the PBAD, P22 PR, and PCMV promoters; the regulatory araC gene; and start codon-modified murA and asdA genes. Additionally, the plasmid incorporates DTS (I), DTS (II), and SV40 late poly(A) signals to support effective transcription and translation. The TiLV DNA vaccine constructs were transfected into the HEK293T cell line and the fish cell line EPC using Lipofectamine 2000, as previously described. The His-tagged TiLV antigens were detected via Western blot analysis using an anti-His-tag antibody, with SuperSignal™ West Pico PLUS Chemiluminescent Substrate employed for signal visualization. Positive signals were observed in both HEK293T and EPC cells transfected with pG8R9050, pG8R9051, pG8R9052, and pG8R9055, confirming successful antigen synthesis in both cell lines. Table 2. (2) Construction ofE. piscicidaDNA vaccine vector strains to deliver TiLV-DNA vaccine and to be characterized for all genotypic and phenotypic traits including lysis, attenuation and immunogenicity. A series of E. piscicida mutant strains derived from J118 have been constructed, an R plasmid-cured derivative of the highly virulent E. piscicida EIB202, which has been sequenced. J118 is sensitive to all antibiotics. Suicide vector technologies were used with pRE112 to insert all the deletion and deletion-insertion mutations in the DNA vaccine delivery vector strain χ16048, which has the following genotype: ?asdA10 ΔPmurA180::TT araC ParaBAD murA ΔPcrp68::TT araC ParaBAD crp ΔPfur170::TT araC ParaBAD fur ΔendA11 ΔfliC20 ΔtrxL20 ?waaI25. This strain displays regulated delayed lysis, regulated delayed attenuation, increased DNA vector survival or stability and reduced pyroptosis/apoptosis. (3) Fully characterize and evaluate the immunogenicity of TiLV-DNA vaccines and their ability to induce protective immunity to TiLV challenge in tilapia. To deliver the TiLV DNA vaccines into tilapia, the constructs pG8R9050, pG8R9051, pG8R9052, pG8R9055, and the vector control pYA4545 were introduced into the RAEV DNA vaccine delivery strain χ16048. The strains were characterized in the presence or absence of arabinose, with results indicating that they were arabinose-dependent. None of the strains survived in the absence of arabinose. The stability of the recombinant plasmids was assessed over approximately 50 generations of bacterial growth in LB medium under both selective (with antibiotics) and non-selective (with DAP supplementation) conditions. All plasmids remained 100% stable in strain χ16048 throughout the 50 generations under both conditions. We have vaccinated tilapia with the RAEV-TiLV DNA vaccine strains χ16048 (pG8R9050), χ16048 (pG8R9051), χ16048 (pG8R9052), χ16048 (pG8R9055), and the control strain χ16048 (pYA4545) using both intracelomic (i.c.) injection and bath immersion methods. The vaccinated fish were subsequently challenged with virulent TiLV. This experiment is ongoing, and the results will be reported in the final progress report.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Rathor, G.S.; Swain, B. Advancements in Fish Vaccination: Current Innovations and Future Horizons in Aquaculture Health Management. Appl. Sci. 2024, 14, 5672. https://doi.org/10.3390/app14135672

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The target audiences for this project are the scientific community with special interests in bacterial and viral pathogens of farmed fish, educators, consumers, producers, potential investors, students, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, four undergraduate students (Vanessa Campodonico, Alexa Sauvagere, Lex Bleckley, and Diya Rana) at the University of Florida have been trained. Vanessa Campodonico is co-authored in one of the papers that were published in the Vaccines Journal, the research was carried out under this project. Now, she is admitted to a Graduate Certificate program in Molecular and Biomedical Sciences at the FIU Herbert Wertheim College of Medicine. How have the results been disseminated to communities of interest?Banikalyan Swain attended and presented an abstract entitled "Design and construction of generalized vaccine-vector system to protect teleost fish against multiple infectious diseases in aquaculture" at the World Aquaculture and Fisheries Conference (Hybrid Event)" held on May 24-25, 2023 in Tokyo, Japan. Banikalyan Swain gave a seminar at the University of Florida, Department of Infectious Diseases & Immunology, College of Veterinary Medicine in the fall seminar series. What do you plan to do during the next reporting period to accomplish the goals?(i) Construction of TiLV DNA vaccines: We will specify additional N- and C-terminal nucleotide sequences to enable the use of specific restriction enzymes to correctly insert each TiLV antigen encoding sequence into the pYA4545 DNA vaccine vector. Each construct will be introduced into EPC or SSN-1 fish cells to verify the synthesis of proteins of the predicted size. We will then introduce each of the DNA vaccine constructs into c16047 (discussed above) to first evaluate for stability of constructs and then use them for vaccination and TiLV challenge studies to identify the best vaccine candidate(s) (ii) We will first evaluate c16047 constructs (carrying TiLV DNA vector) in comparison to a control with the empty pYA4545 vector fully attenuated by both bath and i.c. inoculation of tilapia administered at various doses. We will also determine which induce antibodies directed at the TiLV protein being expressed. We will also use these antibodies to determine which reacts with an extract of the TiLV virus since at present, which proteins are structural and included in virus particles is unknown. This will also identify those proteins involved in virus replication and maturation but not included in the virus. We will also determine whether those antibodies induced by viral structural proteins are capable of neutralizing virus infection in EPC or SSN-1 cells. (iii) We will investigate the ability of the selected c16047(pYA4545-TiLV gene constructs) to induce protective immunity in tilapia to TiLV challenge and generate TiLV neutralizing immunity. All studies will be repeated to ensure being able to reach statistically significant conclusions. Based on the results during the studies of initial studies with tilapia, we will decide whether to construct vaccine strains that deliver the ability to induce immune responses to multiple TiLV proteins. If so, then after making these constructs all studies noted above would be repeated

Impacts
What was accomplished under these goals? (i) There are ten genomic sequences encoding ten proteins in the TiLV genome. We have characterized each protein on its structural aspects (α-sheets, β-barrels, turns, coils, etc.) plus hydrophobicity, surface display, and potential antigenicity (B & T cell epitope prediction) along each molecule. Each gene sequence has been codon optimized and modified to specify the synthesis of a C-terminal His tag sequence to enable monitoring of synthesis of the fusion protein using a monoclonal antibody recognizing the His sequence. Initially, TiLV gene segments were inserted into pcDNA/3.1/A vector. HEK293T cells were transfected with pcDNA/3.1/A-TiLV plasmids using Lipofectamine 2000 and cells were incubated at 37 ?C with 5% CO2. After 24 and 48 hours of transfection, cells were harvested and lysed in a sample loading buffer. Cell lysates were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane for western blot Analysis. Anti-His-tag antibody was used for the detection of the TiLV antigen synthesis. SuperSignal™ West Pico PLUS Chemiluminescent Substrate was used to develop the blot. Cells transfected with pG8R9029, pG8R9030, pG8R9031 and pG8R9035 showed positive signals. (ii) TiLV antigen synthesis in E. coli TiLV gene segments (2 - 10) were codon optimized based on E. piscicida codon usage. Plasmids listed in Table-2, TiLV gene segments were inserted into the pET28a)+ vector at the SalI and SacI restriction enzyme site and the resultant plasmids were named pG8R9020 - pG8R9028. Antigen synthesis was confirmed by western blotting (Fig. 2). (iii) Construction of TiLV plasmid constructs and confirmation of TiLV antigen synthesis in E. piscicida The regulated lysis vectors depicted in Fig.3A & B both have Ptrc-regulated synthesis of protective antigens for delivery by cell lysis and araC ParaBAD-regulated murA and asd genes with GTG start codons to decrease translation efficiency. Recombinant antigen delivery is achieved during lysis of the RAEVs or by action of host phagocytic cells breaking down RAEV cells in the case of using vectors such as pG8R111 (Fig. 3A). Use of pG8R114 (Fig. 3B) vector with the fusion of antigens to the bla SS (T2SS) leads to the delivery of antigens to the periplasm, resulting in an increased production of outer membrane vesicles (OMVs) that enhance immunogenicity and antibody production against delivered antigens. In addition, the use of Type 2 secretion for protective antigen delivery also leads to protective antigens released into the supernatant fluid surrounding cells to enhance the level of induced immune responses. The TiLV gene segments were inserted into plasmid pG8R111 or pG8R114 (Table 2) and the resultant TiLV plasmids were electroporated individually into E. piscicida strain χ16035: DasdA10; ΔPfur170::TT araC ParaBAD fur; DPcrp68::TT araC ParaBAD crp ?relA20::araC ParaBAD lacI TT. Synthesis of TiLV antigens was confirmed by western blotting (FIG. 22A-22B). TiLV antigen synthesis was detected in χ16035(pCHC104) and χ16035(pCHC107) (Fig. 3CB). The plasmid stability of these two strains growing under permissive conditions (presence of arabinose and DAP in media) was studied for up to 100 generations. These results showed that these plasmids were stable in E. piscicida vaccine strains and retained the ability to produce the TiLV antigens. (2) Construction ofE. piscicidaDNA vaccine vector strains to deliver TiLV-DNA vaccine and to be characterized for all genotypic and phenotypic traits including lysis, attenuation and immunogenicity. A series of E. piscicida mutant strains derived from J118 have been constructed, an R plasmid-cured derivative of the highly virulent E. piscicida EIB202, which has been sequenced. J118 is sensitive to all antibiotics. Suicide vector technologies were used with pRE112 to insert all the deletion and deletion-insertion mutations in the DNA vaccine delivery vector strain χ16047, which has the following genotype: ?asdA10 ΔPmurA180::TT araC ParaBAD murA ΔPcrp68::TT araC ParaBAD crp ΔPfur170::TT araC ParaBAD fur ΔendA11 ΔtrxL20 ΔfliC20 ?relA20::araC ParaBAD lacI TT ?waaI25. This strain displays regulated delayed lysis, regulated delayed attenuation, increased DNA vector survival or stability and reduced pyroptosis/apoptosis. These are the required feature for an ideal DNA vaccine delivery system. (3) Fully characterize and evaluate the immunogenicity of TiLV-DNA vaccines and their ability to induce protective immunity to TiLV challenge in tilapia. For the injection groups, tilapia in the three groups (119.81 ± 53.21 g; 16.41 ± 2.28 cm) exhibited clinical signs of E. piscicida infection between 2-22 days post-exposure, including exophthalmia and cloudy eyes, external hemorrhages of the skin, mild to moderate dermal ulceration, lethargy and anorexia, and enlarged gall bladder (Figure 4). Mortality began at 2 days post-exposure and continued until day 22 with cumulative mortality of 40% (4/10), 30% (3/10), and 60% (6/10) in the 103, 104, and 105 CFU/ml infection dose groups, respectively (Figure 5). In the control group, 3 out of 10 fish were euthanized due to aggression, and it was confirmed that these fish were negative for E. piscicida by bacterial culture. E. piscicida was isolated from the kidney cultures of the fish that showed clinical signs. Contrarily, all survivor fish from the three treatment groups were negative for E. piscicida using the same bacterial culture protocols.The Tilapia fish that were exposed to E. piscicida through bath immersion (98.71 ± 34.25 g; 16.28 ± 1.94 cm) showed similar signs of infection as the fish that were injected with the same bacteria (Figure 6). The onset of mortality began on day 6 and continued until day 17, with a cumulative mortality rate of 10% (1/10), 10% (1/10), and 30% (3/10) in the groups that were exposed to infection doses of 104, 106, and 108 CFU/ml, respectively (Figure 7). In the control group, 2 fish out of 10 were euthanized due to aggression, but bacterial culture confirmed that they were not infected with E. piscicida. Kidney cultures of the infected fish showed the presence of E. piscicida, whereas all surviving fish in the three groups were found to be negative for the bacteria using the same bacterial culture protocols.The LD50 value for injection was calculated by using the AAT Bioquest (2023) calculator and resulted in an LD50 of 45,568.206 CFU/ml (Figure 8). However, the LD50 value for bath immersion could not be calculated as the highest mortality rate generated was 30%. Therefore, the LD50 value for bath immersion must be higher than the highest concentration administered in this study (108 CFU/ml).

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Swain B, Campodonico VA, Curtiss R 3rd. Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish Against Edwardsiellosis. Vaccines. 2023 Sep 9;11(9):1470. doi: 10.3390/vaccines11091470. PMID: 37766146; PMCID: PMC10534663.