Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: advantages for immunization strategies and vaccine implementation.

Rabbit hemorrhagic disease virus (RHDV) VP60 capsid protein was recently expressed at approximately 1.5 gL(-1) associated with the disruption pellet of Pichia pastoris, thus requiring an additional process of extraction by solubilization. Consequently, the expression of a soluble variant of VP60 was undertaken in order to attain an easier approach for vaccine production. The VP60 gene was cloned without secretion signal under the transcriptional control of the AOX1 yeast promoter. The antigen obtained was intracellular and soluble at approximately 480 mg L(-1). Its characterization by size-exclusion HPLC, ultracentrifugation, and electron microscopy, showed the presence of high molecular weight structures similar in mass, size and buoyant density to native RHDV. The antigenic profile was similar to that from authentic virions as determined with monoclonal antibodies directed against RHDV conformational epitopes. These analyses, conducted on VP60 obtained insoluble in P. pastoris revealed the formation of protein aggregates rather than the presence of ordered multimeric structures. An immunization trial was conducted in which the soluble VP60 was employed by subcutaneous (s.c.) injection either purified by a single chromatographic step or contained within raw disruption supernatant, emulsified in Montanide 888. The insoluble variant was administered as a yeast extract powder by oral and s.c. routes. The earliest IgG response, titers and persistence of antibodies were studied by competition ELISA and hemagglutination inhibition (HI) assays. All rabbits immunized with the yeast-derived antigens developed a strong RHDV-specific response (including the "RHDVa" subtype) that lasted over one year after the primary immunization. Early HI titers up to 1/40 960 were generated. The immune response was similar to that induced by VP60 from Sf9 cells and superior to the response elicited with inactivated RHDV. Overall it was found that the soluble VP60 multimers, safely and easily produced in P. pastoris, are a valuable candidate for the rational implementation of a low-cost, scalable subunit vaccine against RHDV.