Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate

Abstract

Background: Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine.

Results and conclusion: We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

Keywords: Artificial recombinant protein; B cell epitope; FMDV; GH loop; Multi- epitope.

Fig. 1

Phylogenetic tree via the correlation analysis of seventy-one GH loop sequence (reside 136–162) from FMDV type O VP1 protein. Height, y axis means the number of different amino acids among GH loops. Open square boxes mean variant cluster and arrows indicate five sequences incorporated in 5BT design

Fig. 2

a Schematic diagram for construction of recombinant proteins expression vector system. b SDS–PAGE gel stained with Coomassie Brilliant Blue. Each lane was loaded with 2 µg of purified recombinant proteins. Lanes: M, proteins marker; 1, 5BT (18.1 kDa); 2, B5BT (45.8 kDa); 3, BmpB (28.7 kDa); 4, BSA (66.5 kDa). c Western blot analysis recombinant proteins were detected with His-tag using anti-His-tag antibody. BmpB is a positive control and commercial BSA is a negative control

Fig. 3

SDS–PAGE analysis for the expression pattern of recombinant proteins. Lanes: M, protein marker; S, soluble crude protein; IB, crude inclusion bodies; 5BT, 18.1 kDa; B5BT, 45.8 kDa; BmpB, 28.7 kDa. The stained gel was analyzed by image J software and inclusion body fractions were: 5BT, 36%; B5BT, <2%; BmpB, <1%

Fig. 4

BmpB effect on the stability of recombinant proteins. a SDS–PAGE gel stained with Coomassie Brilliant Blue. Crude protein extracts were incubated at 4 °C for 6 days and daily sample was analyzed by SDS–PAGE. Lanes: M, protein marker. b Graph showing intact proteins in (a) analyzed by image J software

Fig. 5

Validation of recombinant proteins as FMDV subunit vaccine in vivo. a Schematic view of immunization and blood sapling schedule (n = 5/group). b Antibody titer analysis by ELISA. Specific antibody titers against 5BT was measured by ELISA in serum samples collected at days 0, 13, 27 and 42 post-immunization. Antibody titers were expressed as the reciprocal log10 of the last dilution calculated by interpolation to give an absorbance of 1 above background. Each point corresponds to the geometric mean of each groups. Error bars represent standard error of the mean. Validation of immune response route of recombinant proteins. c 5BT specific total IgG titers detected by ELISA at day 42 post-immunization, d IgG1, and e IgG2a. Endpoint titers were expressed as the reciprocal log10 of serum dilutions. Each symbol represents the value of individual mouse. Horizontal lines indicate the mean of each group of animals

Fig. 6

a Detection of FMDV specific antibody response in serum analyzed by competition ELISA assay at day 42 post-immunization. The PI (%) means the percent inhibition. [PI = 100–100 × (OD₄₅₀ of sample serum/OD₄₅₀ of negative control)]. Negative and positive controls were satisfied with standard recommending of manufacturer manual. Each symbol represents the value for individual animals. Horizontal lines describe the mean value for each group of animals. **: P < 0.01; ***: P < 0.001, one way ANOVA. b Antibody response to each peptide in 5BT in serum analyzed by ELISA assay at day 42 post-immunization. Antibody titers were expressed as the reciprocal log10 of the last dilution calculated by interpolation to give an absorbance. Error bars represent standard deviation