A multi-epitope plant-made chimeric protein (LTBentero) targeting common enteric pathogens is immunogenic in mice

Abstract

A plant-based multiepitopic protein (LTBentero) containing epitopes from ETEC, S. typhimurium, and V. parahaemolyticus was produced in plants cells and triggered systemic and intestinal humoral responses in immunized mice. Around 200 million people suffer gastroenteritis daily and more than 2 million people die annually in developing countries due to such pathologies. Vaccination is an alternative to control this global health issue, however new low-cost vaccines are needed to ensure proper vaccine coverage. In this context, plants are attractive hosts for the synthesis and delivery of subunit vaccines. Therefore, in this study a plant-made multiepitopic protein named LTBentero containing epitopes from antigens of enterotoxigenic E. coli, S. typhimurium, and V. parahaemolyticus was produced and found immunogenic in mice. The LTBentero protein was expressed in tobacco plants at up to 5.29 µg g^-1 fresh leaf tissue and was deemed immunogenic when administered to BALB/c mice either orally or subcutaneously. The plant-made LTBentero antigen induced specific IgG (systemic) and IgA (mucosal) responses against LTB, ST, and LptD epitopes. In conclusion, multiepitopic LTBentero was functionally produced in plant cells, being capable to trigger systemic and intestinal humoral responses and thus it constitutes a promising oral immunogen candidate in the fight against enteric diseases.

Keywords: Antibody response; Enteric diseases; Multiepitopic; Plant-based vaccine; Recombinant protein.

Fig. 1

Map of the expression vector used to produce the LTBentero multiepitopic protein in plants. The LTBentero gene comprises the signal peptide from the Glycine max vegetative storage protein, the full-length mature sequence of the E. coli heat-labile enterotoxin B subunit, a four aminoacid linker, followed by the epitopes from antigens ST, FliC and LptD and the SEKDEL endoplasmic reticulum (E.R) retention signal. The LTBentero-coding gene was cloned into the pBI121 vector to drive its expression under the constitutive 35SCaMV promoter. The vector possesses the nptII as gene marker that confers kanamycin resistance in the transformed plants

Fig. 2

Aspect of the tobacco cultures during the plant transformation process with the LTBentero gene. a Calli. b Regenerated shoots at 6 weeks post-infection. c Shoots in rooting medium. d Growing plants under greenhouse conditions

Fig. 3

LTBentero transgene and protein detection. a PCR analysis was conducted using genomic DNA extracted from putative transgenic or WT tobacco plants and primers landing at the 35S promoter and the NOS terminator. b Dot Blot analysis using leaf protein extracts from transgenic or WT plants using a mouse anti-ST serum to confirm LTBentero production. c Western blot analysis using a mouse anti-LTB serum to assess the presence of the LTBentero protein

Fig. 4

Accumulation levels of the LTBentero protein in transgenic tobacco plants determined by ELISA. Immunodetection was performed in leaf protein extracts from transgenic plants or WT plant as a negative control using a mouse anti-LTB serum to quantify LTBentero. A standard curve made with pure LTB was used to determine the levels of the recombinant protein in each line

Fig. 5

Systemic IgG antibody response induced in mice by the LTBentero plant-made protein. a anti-LTB b anti-ST and c anti-LptD antibody titers in subcutaneously (s.c.) or orally (per os, p.o.) immunized mice. Antibody levels were determined by ELISA weekly over a period of 4 weeks post-first immunization.

Fig. 6

Mucosal IgA antibody response induced in mice by the LTBentero plant-made protein. a Anti-LTB and b anti-ST antibody titers in subcutaneously (s.c.) and orally (p.o.) immunized mice. Antibody levels were determined by ELISA weekly over a period of 4 weeks post-first immunization

Fig. 7

Mucosal IgA antibody response induced in mice by the LTBentero plant-made protein. Anti-LptD and anti-FliC antibody titers in orally-immunized mice. Antibody levels were determined by ELISA weekly over a period of 4 weeks post-first immunization