Strategies and Limitations in Dendrimeric Immunogen Synthesis. The Influenza Virus M2e Epitope as a Case Study

Abstract

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.

Peptide vaccine candidates against classical swine fever virus: T cell and neutralizing antibody responses of dendrimers displaying E2 and NS2–3 epitopes

“…The chemical ligation at pH 7 was monitored by HPLC and MALDI‐TOF MS. Several drawbacks were found with this method because the linear peptides were difficult to obtain in desirable amounts and purity, and even when available they were quite insoluble in solvents useful for the conjugation process. The difficulties encountered in these three syntheses are not infrequent49, and in our case they eventually led to the convergent approach being discarded in favor of stepwise SPPS (Figure 1, right panel). Previous work in our laboratory had demonstrated that the introduction of flexibilizing spacer units (e.g.…”

Gold Nanoparticle–M2E Conjugate Coformulated with Cpg Induces Protective Immunity Against Influenza A Virus

“…Other antigen carriers such as keyhole limpet hemocyanin [8], human papilloma virus [9], bacteria [10,11], liposomes [12], virus-like particles [13] and flagellin [14] have been tested. In another strategy, M2e has been developed as a dendrimer containing multiple branched copies of M2e [15,16]. However, many of these studies have used potent yet potentially toxic adjuvants, including derivatives of cholera toxins or heat labile endotoxin, saponin QS21, keyhole limpet hemocyanin and Freund’s adjuvant [8,10,17,18].…”

A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate

“…1). We included a proline residue at position 25 to improve the synthesis of M2e peptide [36][37][38]. We have previously shown [32,34] that a branched lipopeptide in which Pam2Cys, a potent agonist [39] for Toll-like receptor-2 (TLR-2), is attached to the -amino group of a lysine residue inserted between the T helper cell epitope (T H ) and target epitope is more immunogenic than lipopeptides with a linear structure.…”