Proc. Natl. Acad. Sci. USA Vol. 86, pp. 9084-9088, December 1989 Biochemistry Vaccine engineering: Enhancement of immunogenicity of synthetic peptide vaccines related to hepatitis in chemically defined models consisting of T- and B-cell epitopes JAMES P. TAM* AND YI-AN Lu The Rockefeller University, 1230 York Avenue, New York, NY 10021 Communicated by Bruce Merrifield, July 10, 1989 ABSTRACT We report the development of two models for entire amino acid sequence of the S protein, an NH2-terminal synthetic hepatitis B vaccines. The models were based on the fragment, the 55-residue pre-S(2), and a 108- to 119-residue multiple antigen peptide (MAP) system and contained the pre-S(1), the length ofwhich depends on the HBV strain. The relevant B- and T-cell epitopes without any macromolecular pre-S(1) region is located upstream from the pre-S(2) region, carrier. Two peptides, representing the a determinant of the S irrespective of subtypes. The virus-neutralizing antibodies region (S protein) of hepatitis B surface antigen, a dominant are elicited by the S region ofthe HBsAg as well as the pre-(S) serotype of hepatitis B virus infection found in humans, and region of the HBV (2-4). residues 12-26 ofthe pre-S(2) region ofthe middle protein were Serologically, HBsAg has one group-specific determinant incorporated as either monoepitope or diepitope MAP models. (a) and two sets of mutually exclusive determinants (d or y Immunizations of outbred rabbits with the monoepitope MAP and w or r), giving four major serotypes (adw, ayw, adr, and that contains the pre-S(2) antigen resulted in high-titered ayr). Bhatnager et al. (5) and others (6, 7) reported that antibody response to the middle protein, but the other mono- residues 139-147 of the S protein contained an essential part epitope, containing only the a-determinant peptide antigen, of the a determinant of HBsAg. Because most anti-HB resulted in poor immune responses to either the peptide response in humans is generally directed against the common antigens or to the S protein. The diepitope MAPs containing determinant a, encountered in all serotypes of HBV, immu- both the a and the pre-S(2) determinants produced high-titer nization with one serotype of HBsAg is protective against all antibodies reactive to the a-synthetic peptide and the S protein, serotypes. Itoh et al. (8) and others (9, 10) reported that a as well as to the middle proteins. Thus, our results show that peptide of the pre-S(2) protein (residues 14-32) raised pro- the diepitope MAP models eliminate the need for a protein tective antibodies in chimpanzees. These synthetic peptides carrier and that the pre-S(2) peptide determinant serves as a were coupled to protein carriers such as keyhole limpet T-helper cell epitope that enhances the immune response of the hemocyanin for immunization. Because protein carriers are S region and overcomes the poor immunogenicity encountered undesirable for human vaccines, we have recently designed with a single epitope of the S region. a chemically defined approach known as multiple antigen peptide (MAP) system to incorporate peptide antigens that A long-term goal for the development of synthetic vaccines give a macromolecular structure without a large protein is the design of chemically unambiguous, multivalent vac- carrier (11, 12). MAPs were sufficiently immunogenic and cines containing the relevant B- and T-cell epitopes but elicit specific monoclonal and polyclonal antibodies to rec- requiring no macromolecular carrier. Such synthetic vac- ognize their cognate proteins. MAPs were also designed to cines would eliminate many irrelevant determinants of bac- allow flexibility to incorporate multiple epitopes in a chem- terial and viral vaccines that may cause undesirable side ically unambiguous system. In this study, we describe the effects. Hepatitis B virus (HBV) represents an important design oftwo chemically defined models that incorporate two target for a synthetic peptide vaccine model because it affects epitopes of HBsAg, the a determinant of the S region nearly 200 million people worldwide, and chronic infection consisting of residues 140-146, and the pre-S(2) antigen produces a risk of developing hepatocellular carcinoma (1). consisting of residues 12-26. Our results show that immuni- The development of a conventional vaccine has been hin- zation of outbred rabbits with the monoepitope models dered by the difficulty of growing HBV in tissue culture. containing only the a determinant peptide antigen produced Currently available vaccines for HBV include subviral com- no to poor immune responses to either the peptide antigen or ponents of inactivated hepatitis B surface antigen (HBsAg) to the HBsAg containing the S region, whereas the diepitope purified from plasma ofasymptomatic HBV-infected carriers models containing both the a and the pre-S(2) determinants and surface proteins obtained from the DNA recombinant produce high-titered antibodies reactive to the synthetic a method. The limitations of both vaccines include production peptide and the HBsAg, as well as to the pre-S(2) peptide costs and safety in the former case and the incomplete antigen. inclusion of all epitopes (e.g., pre-S region) in the latter case (2). A synthetic vaccine based on synthetic peptides may MATERIALS AND METHODS provide an improved design, the necessary supply, and the safety needed for an efficacious vaccine. Synthesis of MAP Containing One Epitope. The synthesis of The DNA of HBV has a large open reading frame encom- MAP was accomplished manually by a stepwise solid-phase passing 1167-1200 base pairs (3, 4). The S gene encodes for procedure (11-13) on t-butoxycarbonyl (Boc)-Ala-OCH2- the S protein, the major antigen ofthe HBsAg, which consists Pam resin (14) (0.1 mmol of alanine is present in 1 g of resin). of 226 amino acid residues. Two minor but larger envelope proteins (the middle proteins) contain, in addition to the Abbreviations: MAP, multiple antigen peptide; HBV, hepatitis B virus; HBsAg, hepatitis virus surface antigen; Boc, t-butoxycar- bonyl; Boc-Lys(Boc), Na,NE-Boc2-Lys; Fmoc, 9-fluorenylmethox- The publication costs of this article were defrayed in part by page charge ycarbonyl; Acm, acetamidomethyl; DCC, dicyclohexylcarbodiim- payment. This article must therefore be hereby marked "advertisement" ide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9084 Downloaded by guest on October 2, 2021 Biochemistry: Tam and Lu Proc. Natl. Acad. Sci. USA 86 (1989) 9085 After removal of the Boc group by 50% trifluoroacetic acid trifunctional amino acids as follows: Glu(tert-butyl ester), and neutralization of the resulting salt by diisopropylethyl- Asp(tert-butyl ester), Lys(Boc), Thr(tert-butyl ester). Depro- amine (DIEA) the synthesis of the first level of the carrier tection by piperidine was preceded by one piperidine pre- core to form [Boc-Lys(Boc)]-Ala-OCH2-Pam resin was wash, and the coupling was mediated with DCC/1-hydroxy- achieved using a 4-mol excess of Boc-Lys(Boc) via dicyclo- benzotriazole in dimethylformamide. After completion of syn- hexylcarbodiimide (DCC) alone in CH2Cl2. The second and thesis, the MAP-resin was treated with low-high HF (15) to third levels were synthesized by the same protocol to give the remove the peptide chains from the resin. The procedure for octabranching Boc-Lys(Boc) core matrix. For synthesizing Fmoc amino acid peptide chain was as follows: (i) 20 ml of the core matrix of a MAP containing two different antigen dimethylformamide (three times for 1 min); (ii) 20 ml of sequences, 9-fluorenylmethoxycarbonyl (Fmoc)-Lys(Boc) piperidine/dimethylformamide (1:1, vol/vol) (1 min); (iii) 20 was used in the third level to give Fmoc-Lys(Boc) end ml of piperidine/dimethylformamide (1:1, vol/vol) (10 min); groups. The protecting group scheme for synthesis ofantigen (iv) 20 ml ofdimethylformamide (three times for 1 min); (v) 20 was as follows: Boc group for the a-NH2 terminus and ml of CH2Cl2 (three times for 1 min); (vi) 20 ml of dimethyl- side-chain protection groups for the trifunctional amino acids formamide (two times for 1 min); (vii) amino acid (4 equiva- were: Glu(benzyl ester), Lys(p-chlorobenzyloxycarbonyl), lents) of 5 ml of dimethylformamide (5 min), 1-hydroxyben- Thr(benzyl), Asp(benzyl ester), Cys(acetamidomethyl; zotriazole (4 equivalents) in dimethylformamide, DCC (4 Acm), Arg(tosyl), and Tyr(p-bromobenzyloxycarbonyl). De- equivalents) in CH2Cl2 were added for 2 hr; (viii) 20 ml of protection by trifluoroacetic acid was preceded by one tri- dimethylformamide (four times for 2 min); (ix) 20 ml ofCH2CI2 fluoroacetic acid prewash. Neutralization by DIEA was in (two times for 2 min). dimethylformamide. The coupling was mediated with DCC/ Dimerization of Two Heterlogous Epitope MAPs by Oxida- 1-hydroxybenzotriazole in dimethylformamide. After com- tion to the Disulfide. To 1 ,umol of MAP, the diepitope pletion of the synthesis, the MAP-resin was treated with containing Cys(Acm) dissolved in a deaerated and N2- trifluoroacetic acid to remove the Na-Boc groups, then purified 50% acetic acid solution at room temperature, 500 Iul acetylated with 10%o acetic anhydride/10To DIEA in CH2CI2, of a solution of 12 in MeOH (1 mM solution) was added and finally cleaved with the low-high HF method (15) to batchwise for 1 hr at 0°C. The reaction was quenched by remove the MAP from the resin support. The crude peptide adding 1 M aqueous sodium thiosulfate until the yellow color was then washed with cold ether/mercaptoethanol (99:1 was removed. MeOH was removed by dialysis in 0.1 acetic vol/vol) to remove p-thiocresol and p-cresol and extracted acid, and the desired MAPs were purified by gel-permeation into 8 M urea in 0.1 m Tris HCI buffer (pH 8.0).