Difference of T cell and B cell activation in two homologous proteins with similar antigenicity but great distinct immunogenicity
Introduction
Hepatitis E virus (HEV) is a major cause of acute hepatitis in the world (Amon et al., 2006, Masuda et al., 2005, Worm et al., 2002). The HEV genome consists of three open reading frames (ORFs), among which ORF 2 encodes the 660 amino acids (aa) length of capsid protein (Emerson and Purcell, 2003). A bacterially expressed particulate hepatitis E vaccine, HEV 239 (ORF2 aa 368–606), has been shown to be highly immunogenic and protect primates against HEV infection (Li et al., 2005a, Li et al., 2005b). Results of phase I and phase II clinical trials showed that this vaccine is safe and efficacious for humans too (Xia et al., unpublished observations). HEV 239 has virtually identical antigenicity as its N-terminal 26 aa truncated protein, E2 (ORF2 aa 394–606), and both proteins occur as homodimers in mildly dissociating condition (Zhang et al., 2001). In neutral solution, however, homodimers of E2 engage in dimeric interaction to form hexamer, whereas those of HEV 239 interact to form 27 nm particles (Li et al., 2005a). Monoclonal antibodies raised against E2 are similarly reactive against the vaccine protein and vice versa, and they distinguished at least six antigenic determinants relating to conformation of the homodimer as well as two linear epitopes (Li et al., 2005b, Zhang et al., 2005). Most of these antibodies cross-react with HEV, effecting immune capture of the virus, and at least two of the antibodies could neutralize infectivity of the virus. This suggests that structure of HEV 239 and E2 may model features of the virus capsid, probably the protrusions projecting from the virus shell. Consequently, both proteins are strongly reactive with acute and convalescent sera from hepatitis E patients. However, HEV 239 is over 200 times more immunogenic than E2 (Li et al., 2005b). The mechanism(s) that accounts for the striking difference in intrinsic immunogenicity between these two antigenic similar proteins has not been determined. Therefore, we have begun to address this issue in this study by investigating the role of antigen-specific T helper (Th) cells and the efficiency at level of B cell activation.
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Mice
Six to eight weeks old female BALB/c mice or athymic (nude) BALB/c mice were provided by the animal center of Xiamen University and the animals were kept under specific pathogen free conditions.
Peptides
A total of 46, 15-mer peptides (P1–P46), which span the entire length of the HEV 239 protein and overlap one another by 10 amino acids, were supplied at 70% purity by AC Scientific, China in lyophilized form. The peptides were reconstituted in dimethylsulfoxide (DMSO) to a concentration of approximately
T cell responses induced by HEV 239 or E2
BALB/c mice were immunized s.c. with 20 μg of the HEV 239, E2 or placebo, mixed with alum adjuvant, respectively. Spleen cells were harvested 2 weeks later and HEV 239-, or E2-, specific T cells were enumerated by IFN-γ-ELISPOT assay (Fig. 1A) and IL-5-ELISPOT (Fig. 1B), using Con A as positive control and BSA as negative control. The results showed that HEV 239 vaccination evoked much stronger antigen-specific Th1 and Th2 response than E2 vaccination (P < 0.01), and the activated T cells
Discussion
Having similar amino acids sequence and virtually identical antigenicity, the particulate HEV 239 is over 200 times more immunogenic than E2 (Li et al., 2005b). In present study, we identified the dominant Th epitope and Tc epitope in HEV 239, these epitopes are also contained in E2. Nevertheless, HEV 239 is efficient in evoking a vigorous and predominantly antigen-specific T cell response, while E2 cannot (Fig. 1). When the antigen-specific Th cells were activated by Th epitope peptides
Acknowledgements
This work was supported by Science and Technology Projects of Fujian Province (No. 2004yz01-1), Program for New Century Excellent Talents in University (NCET) and Science and Technology Projects of Xiamen (No. 3502Z20041008), China. We thank Dr. F.P. Huang for helpful discussion.
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