The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.

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We predicted the localization ofa major hepatitis B surface antigen (HBsAg) determinant within residues [Neurath, A. R., Strick, N. & Oleszko, W. R. (1981)J. ViroL Methods 3,[115][116][117][118][119][120][121][122][123][124][125]. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers, and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzymelinked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 x 105 M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155 and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K > 107 M-'). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.The concept ofusing synthetic peptides for active immunization instead of whole viruses or their components was developed a decade ago (1). Experiments designed to test this concept were limited, because the amino acid sequences of only a few viral proteins were known (2). Considerable progress in peptide synthesis (3) and in deducing amino acid sequence data from viral DNA or RNA sequence data (4-16) allows the concept to be examined on a variety of viral antigens. Indeed, there have recently been many experiments directed toward the development of synthetic vaccines (17)(18)(19)(20)(21)(22). In order to be considered effective in active immunization, synthetic peptides should induce long-lasting immunity comparable to that elicited by the corresponding "natural" antigen. Because direct evidence for such comparison can be obtained only by clinical trials, experimental data suggesting that the results of such trials will be promising are needed. The necessity to evaluate the immunologic crossreactivity, on both humoral and cellular levels, between a synthetic fragment intended for vaccination and the intact organism has been emphasized (2) but only rarely appreciated. The work reported here concerns the specificity of antibodies elicited by a synthetic peptide corresponding to residues 135-155 ofthe hepatitis B surface antigen (HBsAg) protein (23). The results stress the need for such evaluations and indicate that sophisticated mimicking of conformational determinants on the HBsAg protein (24), rather than mere synthesis of peptides corresponding to selected segments of the primary structure ofthe protein, may be required for production of synthetic vaccines. MATERIALS AND METHODSThe peptide having...

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