Expression of the multimeric and highly immunogenic Brucella spp. Lumazine synthase fused to bovine rotavirus VP8d as a scaffold for antigen production in tobacco chloroplasts.

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

Conferencia; PLANT-BASED VACCINES, ANTIBODIES & BIOLOGICS; 2015

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. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus (BRV) is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes making it a good candidate for vaccination. The group A C486 BRV VP8* protein was previously expressed by our group in tobacco chloroplasts, mostly as insoluble aggregates, and we demonstrated that it conferred a strong immune response in female mice. Moreover, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. We have also fused BLS to a shorter fragment of the VP8* protein (VP8d) and expressed the resulting fusion (BLSVP8d) in E. coli. BLSVP8d elicited higher antibody titers when compared either to VP8* alone or to 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, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by expressing the BLSVP8d fusion in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly stable in transplastomic leaves. It readily accumulated 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) (4,85 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. Soluble BLSVP8d was also stable and could even be detected in freeze-dried leaves. Furthermore, the immunogenicity of BLSVP8d was assessed in a laying hen model. Specific IgY antibodies could be detected by ELISA after immunization with either fresh or lyophilized unpurified soluble protein extracts. Overall, expression of BLSVP8d as a fusion protein in tobacco chloroplasts represents an interesting platform for a highly immunogenic injectable, or maybe even oral, VP8* subunit vaccine. Dietary uptake of powdered egg yolk specific IgY antibodies could be exploited in the future as an approach to confer passive protection against rotavirus. This study marks the first time the BLS scaffold was assessed in a recombinant plant system and our findings suggest that freeze-drying, without the need for additional lyoprotective components, of transplastomic leaves expressing other antigenic fusions to the potent immunomodulator BLS can further reduce costs and simplify downstream processing, purification and storage, allowing for more rational injectable or oral immunizations schemes.