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

FEDERICO ALFANO; EZEQUIEL M. LENTZ; BELLIDO, D; DUS SANTOS M.J; GOLDBAUM A; WIGDOROVITZ A.;; FERNANDO BRAVO-ALMONACID

VERONA

Congreso; Plant-based vaccines, antibodies and biologicals; 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 (Lentz et al., 2011). Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. BLS assembles as a remarkably stable dimer of pentamers, with 10 N-terminus sites of linkage. 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 (Bellido et al., 2009), while IgY antibodies against BLSVP8d produced in hens, were able to fully protect mice against challenge with virulent BRV in a dose-dependent-manner. (Bellido et al., 2012) In this work, BLSVP8d fusion was expressed in tobacco (Nicotiana tabacum L. cv. Petit Havana) chloroplasts in order to evaluate the capacity of our molecular farming platform to express the family of antigens expressed with the BLS delivery system