MOLECULAR APPRAOCHES TO THE CONTROL OF ICHTHYOPHTHIRIUS INFECTION

Goals / Objectives
The objective of the proposed research is to develop and test vaccines against Ichthyophthirius multifiliis, an economically important pathogen of freshwater fish. Although I. multifiliis is a protozoan parasite, the approach taken in these studies may be applicable to the development of vaccines against a wide range of microbial pathogens.

Project Methods
Over the past several years, we have identified proteins on the surface of the parasite (referred to as i-antigens) which are involved in protective immunity, and have cloned genes encoding those antigens. We intend to transform the non-pathogenic ciliate, Tetrahymena thermophila, with the i-antigen genes of Ichthyophthirius in order to express these proteins on a large-scale. Because Tetrahymena is a normal part of the flora of aquatic environments, it may also be possible to use the transformed cells as a novel live vaccine. The principal goal of this part of the project will be to synthesize chimeric genes which combine sequence elements that control expression in Tetrahymena, with the coding regions of the i-antigen genes from I. multifiliis, and use the chimeric constructs in transformation studies.

Progress 01/01/99 to 12/31/99

Outputs
Our work is aimed at the development of a vaccine against I. multifiliis, a commercially important pathogen of freshwater fish. Along with its practical importance, Ichthyophthirius provides are very useful experimental system for studying the development of cutaneous immunity in fish. In earlier work, we had identified a class of abundant parasite membrane proteins (i-antigens) as targets of the host immune response, and had indirect evidence for the involvement of these proteins in protective resistance. Our plan was to isolate the genes for these proteins and use them in the construction of a genetic (DNA) vaccine. In the previous period, we had isolated and characterized the gene for a 55 kDa i-antigen from a parasite isolate that belongs to serotype D, which is highly prevalent in nature. Over the past year, we made a completely synthetic version of this gene, substituting the preferred codon usage of channel catfish throughout its length. In doing this, we replaced 18 UAA and UAG "stop" codons in the original sequence (these are used as glutamine codons in I. multifiliis). The synthetic copy, along with two additional versions that lack coding regions (at the N- and C-termini) responsible for targeting the protein to the plasma membrane, were cloned into pcDNA3.1, a eukaryotic plasmid expression vector often used for genetic immunization. We have now begun preliminary vaccine trials with these constructs and are awaiting results. The isolation of the 55 kDa antigen has proven useful in other respects. Comparison of the deduced amino acid sequence of the 55 kDa protein with the sequence of a 48 kDa antigen from a serotypic variant of Ichthyophthirius has allowed us to design PCR primers that permit amplification of i-antigen gene fragments from all parasite isolates obtained to date. These "universal" primers should permit further characterization of these proteins, and should greatly facilitate the design of multivalent vaccines. Finally, vaccine trials with the purified i-antigens has shown strong protection, giving further credence to the idea that a practical vaccine against Ichthyophthirius may be possible.

Impacts
(N/A)

Publications

Gaertig, J., Gao, Y., Tishgarten, T., Clark, T. G. and H. W. Dickerson (1999). Surface display of a parasite antigen in the ciliate Tetrahymena thermophila. Nature Biotechnol. 17, 462-465.
Clark, T.G., Lin, T. L., Jackwood, D. A., Sherrill, J., Lin, Y. and H. W. Dickerson (1999). The gene for an abundant parasite coat protein predicts tandemly repetitive metal binding domains. Gene 229, 91-100.
Dickerson, H.W. and T.G. Clark (1998). Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes. Immunol. Rev. 166, 377-384.