The safety and longevity of DNA vaccines for fish.

A plasmid that contained the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22 degrees. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coli beta-galactosidase (beta-gal), which was in the injected myofibres, was detected in all the fish at 4-21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to beta-gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to beta-gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune-like antibodies to itself or to single- or double-stranded salmon testis DNA. Plasmids can therefore induce long-term foreign protein expression whilst inducing humoral and cell-mediated immunity without autoimmunity or integration in goldfish.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

DNA is as effective as protein at inducing antibody in fish.

Antiviral vaccines are needed for fish. 50 microg plasmid DNA in saline by the intramuscular route and 10 microg beta-gal protein in a commercial oil adjuvant by the peritoneal route induced serum antibody of the same titre and avidity in goldfish. The DNA expressed beta-gal under control of the immediate early promoter/enhancer gene of human cytomegalovirus. Commercial bacterin vaccines are administered to fish by the intraperitoneal route with oil and this route for DNA induced only 2-fold less antibody than DNA by the intramuscular route. Bacterin vaccines and antiviral plasmid DNA could therefore be co-injected into the peritoneum of fish in an oil adjuvant as a single dose.

Characterization of prokaryotic recombinant Aspergillus ribotoxin alpha-sarcin.

The Aspergillus ribonuclease alpha-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its ribosomal RNA substrate is unclear. Here we have compared the cytotoxicity of alpha-sarcin to that of ricin, another catalytic toxin that targets the same rRNA sequence but whose mechanism of cell entry is better understood. Intact ricin binds to cell surface components and enters the cells by receptor-mediated endocytosis, whereas the catalytic polypeptide of ricin (the A chain or RTA) which, like alpha-sarcin, is unable to bind to surface components directly and enters cells by fluid phase uptake. Recombinant alpha-sarcin was produced in Escherichia coli and purified to homogeneity. The protein was soluble, stable and its ability to inhibit in vitro protein synthesis was indistinguishable from that of native alpha-sarcin. Further, recombinant alpha-sarcin had the same in vitro protein synthesis inhibition activity as ricin A chain. The cytotoxicity of alpha-sarcin and ricin A chain to HeLa cells was also the same. The cytotoxicity of alpha-sarcin was due to its RNAase activity rather than to specific membrane effects at the cell surface, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity. One interpretation of the data is that a-sarcin enters mammalian cells in the same way as free ricin A chain.