Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

Abstract

Aim: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design.

Methods: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA.

Results: The results in mice showed that the immu-nogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing.

Conclusion: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic dom-ains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Figure 1

Expression, purification, and Western blot analysis of eight GST fusion proteins containing overlapping preS1 fragments in E.coli BL21 (gold). A and B: Cell lysates and purified fusion proteins subjected to 12.5% SDS-PAGE and stained with Coomassie brilliant blue. C: Protein bands on the duplicate gel of cell lysates electroblotted onto a nitrocellulose membrane for Western blot analysis with mice antisera. Lane M: Molecular weight standard. Lanes 1-8: The samples containing preS1 (21-119), preS1 (21-47), preS1 (34-59), preS1 (48-70), preS1 (60-85), preS1 (71-94), preS1 (86-109) and preS1 (95-119) in a sequent order.

Figure 2

Immunogenicity analysis of different fragments in preS1 (21-119) region in mice by ELISA using eight overlapping GST-preS1 fusion proteins as coated antigens. The mice were immunized with His-preS1 (21-119) fusion proteins at wk 0, 2, 4, 6 and 8, and the antisera were collected the following week.

Figure 3

Identification of the precise immunogenic domains in mice by competitive ELISA. The coated antigens were the five fusion proteins containing different preS1 fragments: preS1 (21-47) (A), preS1 (34-59) (B), preS1 (48-70) (C), preS1 (86-109) (D) and preS1 (95-119) (E), respectively. The mouse antisera colleted at wk 9 were previously incubated with the coated antigens or the other overlapping antigen(s) at a dose of 2 µg, and the samples not treated with antigen(s) were used as controls.

Figure 4

Detection of antigenicity (A₄₅₀) of the eight different preS1 fragments overlapped in preS1 (21-119) region against hepatitis B patients’ serum samples with different infection types by indirect ELISA. ¹The antigenicity of the fragments, preS1 (21-47), preS1 (48-70), preS1 (86-109), preS1 (95-119), and especially preS1 (34-59), decreased significantly in chronic-phase sera (chronic hepatitis B and HCC with hepatitis B samples) in comparison with acute-phase sera (P<0.05). However, 2the antigenicity of the fragments, preS1 (60-85) and preS1 (71-94), in the middle part showed less diversity (P>0.05). Each column represents the mean A value±SE.

Figure 5

Identification of the precise immunogenic domains in humans by competitive ELISA. The coated antigens were the seven fusion proteins containing different preS1 fragments: preS1 (21-47) (A), preS1 (34-59) (B), preS1 (48-70) (C), preS1 (60-85) (D), preS1 (71-94) (E), preS1 (86-109) (F) and preS1 (95-119) (G), respectively. The mixture of sera samples from acute hepatitis B patients was previously incubated with the coated antigens or the overlapping antigen(s) at a dose of 2 µg, and the samples not treated with antigen(s) were used as controls.