SynthesisofaDisulfide-LinkedOctamericPeptideConstruct CarryingThreeDifferentAntigenicDeterminants Goran Kragol, a, * Laszlo Otvos, Jr., a JingQi Feng, a Walter Gerhard a and John D. Wade b a The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA b Howard Florey Institute, University of Melbourne, Victoria 3010, Australia Received 14 February 2001; accepted 30 March 2001 Abstract—In an effort to develop peptide vaccines against the influenza virus, we have successfully synthesized a disulfide-linked octameric homodimer that bears four copies of the influenza virus M2 protein ectodomain as well as two copies each of T-helper cell hemagglutinin epitopes, the I-E d restricted S1 and the I-A d restricted S2 fragments. Peptide attachment was via intermolecular disulfide formation from free sulfhydryl-bearing cysteine derivatives in solution. This reaction was efficient only when the amino- group of the cysteine was Fmoc-protected. # 2001 Elsevier Science Ltd. All rights reserved. As part of an on-going endeavor to develop peptide vaccines against influenza virus, we recently reported the synthesis of branched peptide vaccine prototypes carrying single copies of 24-amino acid residue ectodo- main of the M2 protein, the I-E d major histocompat- ibility complex (MHC) restricted S1 and the I-A d restricted S2 fragments as well as four mannose moieties for improved delivery via mannose-receptors of the antigen presenting cells. 1 In contrast to influenza vac- cine candidates that utilize the antigenic properties of the viral glycoprotein, the ectodomain of the trans- membrane ion-channel protein M2 has the potential advantage of providing highly crossreactive protec- tion. 2,3 As polyvalency, which permits crosslinking of antigen-specific immunoglobulin receptors on B-cells, 4 and presence of strong covalently linked helper T-cell determinants are important features of potent B-cell immunogens, we envisioned a vaccine prototype carry- ing four copies of the M2 ectodomain B-cell epitope and two copies each of the S1 and S2 T-helper cell epitopes. Nevertheless, the assembly of such highly complex and well-defined multimeric peptide constructs that are use- ful for subunit vaccine development requires the use of state-of-the-art solid-phase/solution synthesis. Since four-branched peptide constructs tend to be of higher purity than eight-branched, 5 we decided to assemble tetrameric construct 2 with two copies of M2 and one copy each of the S1 and S2 epitopes. Sub- sequent intermolecular disulfide-bond connection of two terminal cysteine residues would then generate high purity homodimer 7 having the desired octameric structure (Scheme 1). This strategy would also allow a comparison of the immunogenicity of the constructs 2 and 7. Disulfide bridges usually stabilize the three- dimensional structure of extracellular proteins by bridg- ing a single chain or by linking different polypeptide chains, such as occurs with relaxin and insulin. 6 Inter- molecular disulfide linkages have also been successfully used for the conjugation of peptides to carrier proteins or for the preparation of proteins with full enzymatic activity. 7,8 However, because of the high molecular weight of the construct 2 and thus an anticipated high steric hindrance around its backbone that may prevent effective disulfide formation, we assumed that connec- tion of two terminal cysteines would be a crucial step in the final yield of the desired octameric construct 7. This communication reports the successful oxidation of this highly complex artificial protein. 9 For the assembly of the resin-bound construct 1, we used a strategy that includes the sequential and regiose- lective synthesis of M2, S1, and S2 epitopes on the side chains of "-amino groups of the poly(lysine-glycine) backbone during the course of backbone extension on the solid support (Scheme 1). 10 Assembly was accom- plished using continuous flow synthesizer (MilliGen 9050) and Fmoc-PAL-polyethyleneglycol-polystyrene 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00252-9 Bioorganic & Medicinal Chemistry Letters 11 (2001) 1417–1420 *Corresponding author. Fax: +1-215-898-5821; e-mail: kragol@ wistar.upenn.edu