Progress 10/01/05 to 09/30/08
Outputs
OUTPUTS: The primary outputs from our 3-year grant have included 1) banked genomes from 10 independent isolates of Ichthyophthirius multifiliis (the causative agent of white-spot disease in freshwater fish); 2) cloned and characterized sequences for variant genes encoding target vaccine antigens (the so-called immobilization antigens, or i-antigens) from 7 of these parasite isolates; 3) reference antisera that can be used to characterize newly obtained wild-type isolates based on their surface antigen expression; 4) universal PCR primers that can be used to clone and characterize gene homologs of the surface immobilization antigens from essentially any new parasite strain; 5) new protocols for the expression of parasite antigens in Tetrahymena thermophila, a novel platform for recombinant protein manufacture; 6) transformed cell lines of T. thermophila that overexpress recombinant immobilization antigens from Ichthophthirius; 7) transformed T. thermophila cell lines expressing chimeric genes in which individual protein coding domains from 2 immobilization antigen genes from I. multifiliis were mixed and matched; 8) ISCOM-adjuvanted subunit vaccine preparations containing i-antigens from two different I. mulfiliis strains; 9) transformed T. thermophila cell lines that overexpress several additional candidate vaccine antigens from I. multifiliis including a leishmanolysin C1 precursor like protein (the third most common theront-specific protein identified in parasite EST libraries), and, an asparaginyl endopeptidase similar to legumain, a lysosomal cysteine protease involved in tissue invasion in other parasite systems; and, 10) protocols and methods for the isolation and culture of antigen-presenting cells from rainbow trout (Oncorhynchus mykiss) analogous in form and function to the dendritic cells of mammals. This work has involved active collaboration with USDA extension agents in Arkansas (ARS Southern Plains Area), members of the Aquatic Animal Health program at Cornell University in New York State, and USDA-funded faculty members at the University of Georgia. Aside from publications, the principal outcomes of these studies have been disseminated at national and international meetings including the Eastern Fish Health workshop in Charleston, SC (2006) and Gettysburg, PA (2007); the New York Bioscience Summit in Rochester, NY (2006); the PepTalk conference in San Diego, CA (2006) where we received first prize for best meeting poster; the Plant and Animal Genome Conference in San Diego, CA (2006 and 2008); the FASEB Summer Research Conference on Ciliate Molecular Biology in Tucson, AZ (2007); and the NOFFI (Nordic Society for Fish Immunology) Conference in Stirling, Scotland (2007). We anticipate additional talks on these studies to be given at the FASEB Summer Research Conference this coming summer in Saxtons River, VT (2009). PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our long-term goal is the development of an effective vaccine against Ichthyophthirius multifiliis, a protozoan parasite that has major impact on commercial aquaculture worldwide. While Ichthyophthirius is highly pathogenic, animals that survive infection resist subsequent challenge, and a class of abundant parasite surface proteins (the i-antigens) has been identified as potential vaccine candidates. When we began these studies, the degree to which these antigens varied in natural populations was unknown. Furthermore, while injectable vaccines for fish were available, new strategies for vaccine delivery allowing oral or bath immunization were considered highly attractive to the aquaculture industry. To address these issues, we sought to 1) collect new parasite isolates from disease outbreaks in New York and other regions of the country; 2) categorize these isolates with respect to i-antigen serotype; 3) isolate and characterize novel i-antigen genes and produce reference antisera that could be uses in future serotyping efforts; 4) determine the extent of i-antigen variation in natural populations; 5) express recombinant subunit antigens for immunization trials using Tetrahymena thermophila as a production platform; and, 6) evaluate ISCOM and chitosan microspheres as delivery systems for recombinant subunit vaccines antigens administered via oral and/or bath immunization. Universal PCR primers based on conserved sequences at the flanking regions of known i-antigen genes were developed and used to amplify i-antigen sequence variants from 7 (of a total of 10) new parasite strains harvested from different regions of the country. These sequences along with new reference antisera produced against 2 parasite strains revealed extensive variation among the i-antigens expressed by parasite populations in the wild suggesting that recombinant subunit vaccines based on these proteins may be difficult to produce. However, the ability to manufacture a beads-on-a-string-type vaccine containing distinct epitopes from multiple i-antigen genes in a single protein may be possible given the ability to overexpress recombinant (chimeric) i-antigens in the non-pathogenic ciliate, Tetrahymena thermophila. Indeed, the ability to overexpress these and other vaccine candidates (namely, cysteine and aspartyl proteases) in this system has important implications for the use of Tetrahymena as low cost manufacturing platform for aquaculture vaccines in general. While considerable progress was made in characterizing the i-antigens as prospective vaccine candidates and expressing these and other proteins in Tetrahymena, efforts to deliver such antigens via the oral or immersion routes met with limited success. Using an ISCOM-based delivery system in which antigens are trapped in 40 nm cage-like micelles consisting of Quil-A (a saponin), cholesterol, and phosphatidyl choline, we found that ISCOM mixture was toxic to fish when delivered parenterally, and failed to elicit immune protection when administered by bath immersion. Time constraints did not permit the production or testing of chitosan-based microspheres.
Publications
- Formigari, A., Boldrin, F., Santovito, G., Cassidy-Hanley, D., Clark, T.G., and E. Piccinni 2008 (Submitted to Protist). Functional characterization of the 5'-upstream region of the MTT5 gene from Tetrahymena thermophila.
- David L. Straus, M. Mozammal Hossain, and Theodore G. Clark (2009). Copper Sulfate Toxicity to two Isolates of Ichthyophthirius multifiliis. Dis. Aqua. Organ. In Press.
- Boldrin F, Santovito G, Formigari A, Bisharyan Y, Cassidy-Hanley D, Clark TG, Piccinni E. (2007). MTT2, a copper-inducible metallothionein gene from Tetrahymena thermophila. Comp Biochem Physiol C Toxicol Pharmacol. 147:232-40.
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Progress 10/01/06 to 09/30/07
Outputs
In the past year, vaccination trials were initiated using channel catfish immunized with a candidate vaccine antigen against the protozoan parasite Ichthyophthirius multifiliis, the causative agent of "white-spot" disease in freshwater fish. The antigen in this case was a 52 kDa surface glycoprotein (referred to as an i-antigen) produced in recombinant form in the free-living ciliate, Tetrahymena thermophila. Previous studies demonstrated an important role for i-antigens in protective immunity, and Tetrahymena provides a novel, low cost platform for the overexpression of these proteins. The purpose of this study was to test so-called "immune stimulating complexes" (or ISCOMS) in combination with bath immersion as a means of vaccine delivery. When compared with injection of individual fish, bath immersion is far more compatible with commercial aquaculture practices, and based on unpublished work, ISCOMS may be may be an effective vehicle for vaccine delivery by the
immersion route. ISCOMS are produced by sonication of protein antigens with phospholipids and the saponin, Quil-A. These combine to form 40 nanometer cage-like structures in which hydrophobic antigens are embedded. In mammals, antigens delivered with the ISCOM matrix can generate both antibody (IgG and IgA), as well as cytotoxic T-cell-mediated responses, and preliminary studies in fish indicate that this vehicle can deliver vaccines to fish via the skin, gill and gut mucosa (Lin, personal communication). Vaccine trials were undertaken with juvenile channel catfish immunized either by bath exposure or by needle-injection with ISCOM adjuvanted i-antigens. The vaccine was prepared by detergent extraction of Tetrahymena cell lines expressing the 52 kDa protein and incorporation into the ISCOM matrix by sonication. Our preliminary studies have shown that at antigen doses required to elicit protective immunity with other adjuvants, the ISCOM matrix was toxic to fish when administered by
intraperitoneal injection. Furthermore, while not toxic when delivered by bath immersion, the ISCOM-based vaccine failed to elicit protective immunity. We intend to repeat these studies to determine whether different compositions of the adjuvant are more effective and less toxic when administered by these routes. In separate studies, we have cloned and expressed two abundant Ichthyophthirius proteases that may be important vaccine candidates beyond the i-antigens. These are a leishmanolysin C1 precursor like protein (the third most common theront-specific protein identified in I. multifiliis EST libraries), and an asparaginyl endopeptidase similar to legumain, a lysosomal cysteine protease involved in tissue invasion in other parasite systems. Finally, we have begun efforts to identify antigen-presenting cells in fish, which may be a key to improving vaccine efficacy.
Impacts
These studies are intended to identify new candidate vaccines, and methods of vaccine delivery for the prevention of "white-spot disease" in farm-raised fish. This will contribute to CSREES national goals and the priorities of research and extension on a variety of levels. Enhanced production of farm-raised fish will help to ensure an affordable and healthy food supply, and at the same time, benefit the aquaculture industry in the state and country as a whole. Importation of seafood products contributes substantially to the U.S. overseas trade deficit, and the value of increased global competitiveness in this arena is quite clear. Efficacious use of vaccines will help to minimize the current large-scale use of formaldehyde and other toxic agents to control disease, thereby reducing environmental pollution associated with current practices.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
In the past year we have been actively involved in procuring I. multifiliis strains from locations in New York State and elsewhere to determine the relative prevalence of i-antigen serotypes for future vaccine development, and to better characterize the antigens with respect to their variant structures. We obtained 3 new isolates from New York (NY4, NY5 and NY6) along with 4 isolates from outside the state; 3 from Arkansas, and 1 from Peru (Ark1, Ark2, Ark3, and Peru1, respectively). The NY5 and NY6 isolates belonged to a previously identified i-antigen serotype (namely, D), while the others appeared to be novel serotypes. Reference antisera were prepared against isolates NY4 and Ark1. The i-antigens genes from NY4, Ark1 and Peru1 were amplified using degenerate PCR primers that recognize conserved regions at the 5'- and 3'-ends of previously identified i-antigen genes, and the resulting fragments were cloned and sequenced. Comparisons of the deduced amino acid
sequences of these new genes with previously characterized i-antigens provide a much clearer picture of the structures of these proteins and how they vary. Briefly, we found that while all members of the i-antigen family have five repetitive amino sequence domains containing periodic cysteine residues, the first two N-terminal repeats vary considerably more than those at the C-terminal end, which are more highly conserved across serotypes. Variation in primary sequence within the N-terminal repeats is consistent with what we now know are the antigenic sites in the proteins. We have mapped these antigenic sites using a novel expression system based on the free-living ciliate, Tetrahymena thermophila. Transgenic Tetrahymena expressing recombinant i-antigens on their surface are rapidly immobilized using reference (monoclonal) antibodies used to define i-antigen serotypes in Ichthyophthirius. By making chimeric antigens that substitute individual repeats from serologically distinct
i-antigens with one another, we demonstrated that the antigenic domains of these proteins lie within the first, or first and second N-terminal repeats. In the coming year, we plan to fuse the N-terminal repeats from different i-antigens with the idea of making a multivalent antigen that can be used as a combination vaccine in fish. In addition to these studies, we have begun efforts to develop immersion- and oral-based vaccines against I. multfiliis. Transgenic T. thermophila cell lines expressing Ichthyophthirius i-antigens on their surface were extracted with detergent and used to prepare immune stimulating complexes (ISCOMS) with Quil-A (a saponin), cholesterol, and phosphatidyl choline. Such complexes form 40 nanometer cage-like structures in which hydrophobic antigens are embedded. In mammals, antigens delivered with the ISCOM matrix can generate both antibody (IgG and IgA), as well as cytotoxic T-cell-mediated responses, and preliminary studies in fish indicate that this vehicle
can deliver vaccines to fish via the skin and gut mucosa. Vaccine trials with juvenile channel catfish immunized by bath exposure and by needle-injection with ISCOM adjuvanted i-antigen are currently in progress.
Impacts
These studies are intended to identify new candidate vaccines, and methods of vaccine delivery for the prevention of 'white-spot disease' in farm-raised fish. This will contribute to CSREES national goals and the priorities of research and extension on a variety of levels. Enhanced production of farm-raised fish will help to ensure an affordable and healthy food supply, and at the same time, benefit the aquaculture industry in the state and country as a whole. Importation of seafood products contributes substantially to the U.S. overseas trade deficit, and the value of increased global competitiveness in this arena is quite clear. Efficacious use of vaccines will help to minimize the current large-scale use of formaldehyde and other toxic agents to control disease, thereby reducing environmental pollution associated with current practices.
Publications
- No publications reported this period
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