Progress 01/01/00 to 12/31/00
Outputs
Efforts are being made to determine whether genetic immunization with plasmid-based DNA vaccines can elicit cutaneous immunity in fish. To test this idea, vaccines are being generated against Ichthyophthirius multifiliis, a parasitic protozoan that is restricted to the skin and gill epithelia of host species. In previous studies, we had identified protective antigens associated with the parasite as a class of abundant surface membrane proteins known as immobilization antigens (or i-antigens). The gene encoding one such antigen (a ~52 kDa protein from the G5 parasite strain) had been cloned and characterized, and a synthetic copy of this gene (in which the native sequence was replaced by the preferred codon usage of channel catfish) has now been made. Different versions of the synthetic gene (encoding either membrane-bound, secreted, or intracellular forms of the protein) were cloned into the plasmid expression vector, pcDNA3.1, and the resulting constructs injected
into juvenile channel catfish. Detectable antibody responses to the vector-encoded antigens were seen in each case, and modest protection was achieved in the face of a lethal parasite challenge. We have recently linked this gene to the coding sequence of the C3d component of trout complement with the idea that the protein conjugate will show enhanced immunogenicity. Finally, in addition to the gene for the ~52 kDa protein, several other i-antigen genes have now been cloned and characterized. Comparisons of the amino acid sequences predicted by these genes have revealed the presence of conserved structural elements within the i-antigens in general that, in turn, have permitted amplification of coding sequences for i-antigens of disparate parasite strains by PCR. Synthetic copies of these genes are currently being assembled.
Impacts
Ichthyophthirius is one of the most important pathogens of freshwater fish and has substantial impact on commercial aquaculture worldwide. An effective vaccine against this agent would increase production efficiency of farm-raised fish and at the same time eliminate the use of chemicals that can contaminate surface and groundwaters with residues that are potentially harmful to human health.
Publications
- No publications reported this period
|
Progress 01/01/99 to 12/31/99
Outputs
Our work is aimed at the development of a vaccine against Ichthyophthirius 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
As the causative agent of "white-spot" disease in freshwater fish, Ichthyophthirius multifiliis kills large numbers of freshwater fish has a substantial impact on commercial aquaculture worldwide. Our work is directed toward the development of an effective vaccine against the pathogen, and may therefore provide economic benefit to the aquaculture industry. Furthermore, an effective vaccine would obviate the need for chemical treatments (which are currently in use, and which have the potential for contaminating surface and ground waters).
Publications
- 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.
- 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.
|
|