Appraisal and prospects of a dimeric synthetic peptide coupled with tetanus toxoid for a bifunctional vaccine against hepatitis B virus infection.

Recovery from a hepatitis B virus (HBV) infection or vaccination with hepatitis B surface antigen (HBsAg) leads to a physiologic immune response specific for the alpha determinant, common to all subtypes of HBsAg. In contrast, an absence of immune response to the alpha determinant is characteristic of persistent HBV infection as well as of the nonresponders to vaccination. Therefore, our goal is to characterize the alpha determinant for synthesis of a bifunctional vaccine which may be useful for active immunization as well as for the safe termination of immune tolerance to HBsAg in carriers. Because the immunochemical activity of the alpha determinant of HBsAg is dependent upon cysteine and lysine residues that are localized in the hydrophilic stretch between amino acid sequence 121-160, we synthesized the following peptide analogues of HBsAg (HBsPA): 122-137, 128-134, 139-147, 139-158, 140-158, 145-158 and 150-158. Serologic inhibition of human antibodies against the alpha determinant indicated the antigenicity of the HBsPAs containing the Cys-Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys sequence. After coupling with keyhole limpet hemocyanin (KLH), carrier-peptide conjugates induced in rabbits anti-HBs which was neutralized equally by eight different serotypes of HBsAg. Therefore, HBsPA/139-147 represents an essential part of the alpha determinant. By substituting alpha-aminobutyric acid (Aba) for Cys at residue 147 a homogeneous dimeric form of this nonapeptide was prepared. After coupling with purified tetanus toxoid or KLH as a carrier by means of carbodiimide, the product induced sustained high level anti-HBs/alpha response in carrier-primed rabbits. Experiments in chimpanzees should validate our concept of a bifunctional synthetic vaccine against HBV infection.