Expression of VP8d fused to Brucella spp. Lumazine synthase in tobacco chloroplasts as a low-cost platform for highly immunogenic antigen production

E. FEDERICO ALFANO; EZEQUIEL M. LENTZ; DEMIAN BELLIDO; MARIA JOSÉ DUS SANTOS; FERNANDO GOLDBAUM; ANDRÉS WIGDOROVITZ; FERNANDO BRAVO-ALMONACID

Verona

Conferencia; PLANT-BASED VACCINES, ANTIBODIES & BIOLOGICS; 2013

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens. The C486 BRV VP8* protein has been expressed in tobacco chloroplasts, mostly as insoluble aggregates, and it has already been demonstrated that it confers strong immune response in female mice. Moreover, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previously evaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. More specifically, BLS has been fused to VP8* protein and expressed in E. coli. The resulting fusion (BLSVP8d) has already been demonstrated to elicit higher antibody titers than VP8* alone or a mixture of VP8* and BLS, both in female mice and in laying hens models. In the first case, suckling mice born to immunized dams were also protected against oral challenge with virulent rotavirus, while IgY antibodies against BLSVP8d produced in hens, were able to fully protect mice against challenge with virulent BRV in a dose-dependent-manner. In this work, BLSVP8d fusion was expressed in tobacco chloroplasts. This fusion protein was highly stable in transplastomic leaves. It readily accumulates in young leaves and, owing to its stability, the highest expression levels were observed in old leaves, accounting for at least 40% of total soluble protein (TSP) (4,71 mg/g fresh tissue), which was roughly 10 times the expression levels observed for previous VP8* transplastomic plants. The fusion protein remained mainly soluble despite the high expression levels and the fact that VP8* formed inclusion bodies in tobacco when expressed alone. Furthermore, BLSVP8d could be detected even in senescent and dried leaves. This last property, coupled to the high expression levels, contributes greatly to its processing, purification and storage. Its immunogenicity remains to be assessed in a laying hen model. Overall, expression of BLSVP8d as a fusion in tobacco chloroplasts presents interesting advantages. It represents a low-cost platform for a readily purificable and highly immunogenic injectable, or maybe even oral, VP8* subunit vaccine.