Screening and identification of B-cell epitopes within envelope protein of tembusu virus

Abstract

Background: Tembusu virus is a newly emerging flavivirus that caused egg-drop syndrome in ducks in China. TMUV envelope protein is a major structural protein locates at the surface of tembusu virus particle. During tembusu virus infection, envelope protein plays a pivotal role in induction of neutralizing antibody. However, B cell epitopes within envelope protein have not been well studied.

Method: A series of 13 peptides derived from E protein of tembusu virus were synthesized and screened by Dot blot with tembusu virus-positive duck serum. Potential B-cell epitopes were respectively fused with GST tag and expressed in E. coli. The immunogenicity and protective efficiency of epitopes were assessed in ducks.

Results: Dot blot assay identified the peptides P21 (amino acids 301-329), P23 (amino acids 369-387), P27 (amino acids 464-471) and P28 (amino acids 482-496) as potential B-cell epitopes within the envelope protein of tembusu virus. Immunization of prokaryotically expressed epitopes elicited specific antibodies in ducks and the specific antibody elicited by P21, P27 and P28 could neutralized tembusu virus. In addition, protective test suggested that P21 and P27 could completely protect immunized ducks from TMUV challenge.

Conclusion: Four potential B cell epiotpes within tembusu virus envelope protein were identified and analyzed in vitro and in vivo. It was demonstrated that two of them (P21 and P27) could elicit neutralizing antibodies in ducks and offer complete protection against tembusu virus challenge. This findings will contribute to the development of epitope vaccine for tembusu virus prevention.

Keywords: B-cell epitope; Envelope protein; Neutralization; Tembusu virus.

Fig. 1

Identification of the B cell epitope by duck anti-TMUV serum. a Synthesized peptides were probed for reactivity with duck anti-TMUV serum by Dot-blot. Entire E protein and YIRTPACWD were used as positive and negative controls, respectively. b Structure of TMUV E protein showing the position of epitopes. E domains I, II and III were shown in red, yellow and blue. Stem and transmembrane domain were shown in purple and green

Fig. 2

Detection of purified fusion proteins by Western blot using duck anti-TMUV serum. Lane M, molecular weight marker; Lane 1, P21; Lane 2, P23; Lane 3, P27; Lane 4, P28; Lane5, pGEX-4 t-1 vector

Fig. 3

Antibody titer of ducks immunized with B cell epitopes. Groups of 9-day-old duck were immunized twice with P21, P23, P27, P28, entire E protein and PBS by intramuscular injection at 2 weeks interval. Serum samples were collected at 2 weeks after last boost and antibody titer was assessed by indirect ELISA

Fig. 4

Neutralizing activity of anti-epitope antibodies against TMUV. Diluted sera were used to test the neutralizing activity against TMUV by plaque reduction neutralization assay. Data were presented from three independent experiments and statistic analysis was done with SPSS software

Fig. 5

Protective efficacy against TMUV challenge in ducks. Six groups of ducks were inoculated intramuscularly with 10⁴ TCID₅₀ of TMUV. Serum samples were collected daily and inoculated onto BHK-21 monolayer. After 3-day incubation, cell cultures were harvested for detection of TMUV by qPCR. The protection index (PI) was calculated to present protective efficacy of each epitope