Evaluation of mucosal immunity in rainbow trout (Oncorhynchus mykiss) challenged with infectious hematopoietic necrosis virus (IHNV) and the characterization of a recombinant baculovirus-derived IHNV vaccine.

摘要

Cutaneous and lower gastrointestinal (GI) tract mucus of rainbow trout (Oncorhynchus mykiss) were evaluated for immunological responses following exposure to IHNV. Experiments revealed antiviral activity in the GI tract of fish, but the development of specific antibodies (Abs) in cutaneous or GI tract mucus was lacking. Histological examination revealed moderate inflammatory responses and minor focal areas of cellular necrosis in the mucosal epithelial layer of the skin. Virus antigen in numerous tissues and organs was visualized by immunohistochemistry. Results from these experiments indicate a general lack of humoral based immunity to IHNV, but reveal potential innate mechanisms of viral resistance which may provide an important first line of defense against IHNV.; A recombinant IHNV glycoprotein (G protein), expressed in insect cells using a baculovirus vector, was characterized for use as an IHNV vaccine. High levels of protein, slightly lower in molecular mass (M{dollar}sb{lcub}rm r{rcub}){dollar} than wild-type IHNV G, were expressed in Spodoptera frugiperda (Sf9) cells. M{dollar}sb{lcub}rm r{rcub}{dollar} differences were due to glycosylation differences, and it was found that surface expression occurred only when the recombinant glycoprotein was expressed at 20{dollar}spcirc{dollar}C (RecG{dollar}sb{lcub}rm low{rcub}).{dollar} In contrast, expression at 27{dollar}spcirc{dollar}C (RecG{dollar}sb{lcub}rm high{rcub}){dollar} occurred intracellularly, suggesting that at the two temperatures conformational and/or glycosylation differences exist. These results may prove critical in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish.; When RecG{dollar}sb{lcub}rm low{rcub}{dollar} and RecG{dollar}sb{lcub}rm high{rcub}{dollar} were evaluated for immunity in fish, it was found that both were capable of eliciting neutralizing activity in serum of adult rainbow trout, and RecG{dollar}sb{lcub}rm low{rcub}{dollar} was capable of providing limited protection from IHNV challenge. Polyclonal trout anti-IHNV serum possessed Abs that recognized both RecG{dollar}sb{lcub}rm low{rcub}{dollar} and RecG{dollar}sb{lcub}rm high{rcub}{dollar} only as denatured linearized proteins, while Abs from fish immunized with RecG{dollar}sb{lcub}rm high{rcub}{dollar} recognized both the denatured and non-denatured IHNV glycoprotein. This study provides further clarification of a recombinant IHNV glycoprotein produced in insect cells, and emphasizes the need for an increased understanding of the complex interactions of this glycoprotein with the fish's immune system.