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Biotecnología Aplicada

On-line version ISSN 1027-2852

Abstract

FARNOS, Omar et al. Virus-like particles of the Rabbit Hemorrhagic Disease Virus obtained in yeast are able to induce protective immunity against “classical” strains and a viral subtype circulating in Cuba . Biotecnol Apl [online]. 2009, vol.26, n.3, pp.260-266. ISSN 1027-2852.

Four epizootics caused by rabbit hemorrhagic disease virus (RHDV) have been recorded in Cuba from 1993 to 2005. Each time, thousands of animals have died or have been slaughtered to avoid the spread of the disease. Cell culture systems allowing the in vitro replication of RHDV are not available to date. Moreover, the amount of the recombinant capsid protein (VP60) obtained in heterologous expression systems does not commonly exceed a few tens of milligrams per liter of culture. In this paper, we report the expression of VP60 in two strains of the Pichia pastoris yeast with the highest expression levels obtained so far. VP60 was glycosylated, associated to the yeast cell disruption pellet at a concentration of 1.5 g/L in the first case, or soluble in the intracellular fraction at approximately 300 mg/L, following a different cloning strategy. These recombinant variants showed similar antigenic properties to those of the native protein, as determined by monoclonal antibodies. The soluble VP60 showed a higher number of protective epitopes, due to the formation of multimers that were similar in size and structure to the native RHDV capsids. Both antigens induced potent RHDV-specific immune responses in experimental animals. The antibodies produced were able to inhibit the in vitro hemagglutination of a viral strain isolated during the last outbreak in Cuba. A molecular and antigenic characterization of this strain was also carried out and led to its classification as a member of the highly pathogenic RHDVa subtype. Both recombinant antigens induced a specific, protective and long-lasting immune response against “classical” strains and also against the RHDVa subtype.

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