stable scaffold structures could induce antibody responses that target the 2F5-bound peptide conformation [27]. However, to date, no studies have demonstrated induction of gp41 fusion intermediate antibodies by Env or peptide immunogens. In this study we report that a prime-boost regimen of oligomeric gp140 protein and MPER peptide-liposomes was required to induce MPER binding serum responses specific for the 2F5 core gp41 epitope 664DKW amino acids in both guinea pigs and rhesus macaques. The induced antibodies in Rhesus macaques bound the DKW sequence of the nominal 2F5 epitope, and to a protein construct designed to mimic a gp41 fusion intermediate confor- mation. Results Immunogenicity of gp140 oligomer protein and MPER peptide liposomes immunogens in guinea pigs Broadly neutralizing gp41 MPER mAbs 2F5 and 4E10 either do not bind or bind poorly to many Env gp140 oligomers [14,18]. We have previously reported that MPER epitopes are indeed exposed in JRFL gp140CF Env protein allowing binding of 2F5 and 4E10 mAbs [18]. We, therefore, first immunized guinea pigs to determine if either JRFL gp140CF or MPER peptide-liposome complexes that both bind 2F5 and 4E10 mAbs Induction of MPER Antibodies in Rhesus could induce antibody responses to the MPER neutralizing epitope 664DKW. Immunization of JRFL gp140CF oligomer protein alone (36) induced low levels of weak avidity MPER specific responses as indicated by the 100-fold faster dissociation rate (kd) when compared to that of 2F5 mAb (Figure 1A&C). On the other hand, MPER peptide-liposome complexes immunized alone in guinea pigs induced relatively higher avidity antibodies as judged by relatively slower dissociation rates (,6-fold faster in comparison to that of 2F5 mAb) (Figure 1B&D). Thus, compared to Env JRFL gp140CF protein, MPER peptide liposomes induced higher avidity 2F5 peptide-specific responses. Next we determined the fine specificity of MPER specific responses induced by these two immunogens alone. Only guinea pig 1402 when immunized with JRFL gp140CF gave sufficiently high MPER responses. In serum from animal 1402, JRFL gp140CF immunization induced responses targeting 666W residue and an additional residue (669L) outside the 2F5 core (Figure 1E) that is a critical residue for a non-neutralizing MPER mAb [28]. The MPER peptide-liposome immunizations induced responses targeting residues 664D in two animals (1702 and 1703), 665K in one animal (1704) and both 664D and 665K in one animal (1705, Figure 1E). Thus, neither JRFL gp140CF nor MPER peptide- liposomes elicited responses with specificity focused entirely on the Figure 1. Comparison of gp41 MPER specific antibody responses induced in guinea pigs by JRFL gp140CF and MPER peptide- liposomes. The immunization scheme of the study is shown in the top panel. A–B: The 2F5 epitope peptide (SP62 peptide) specific responses in guinea pigs sera (at 1:50 dilution) determined by SPR are shown after vaccination with JRFL gp140CF (A) and MPER peptide-liposome (B). PB, pre- bleed; P1–P3, post-immune bleeds 1–3. C–D: SPR sensogram displaying the comparison of 2F5 epitope peptide specific binding of guinea pig 1402 (C) and 1703 (D) sera at different time points with 2F5 mAb is shown as representative data. The estimated dissociation rates (kd) from the sensogram are indicated for 2F5 mAb and Post-bleed 3 (P3). E: Epitope mapping of post-immune bleed 3 of guinea pigs 1402 (immunized with JRFL gp140CF alone) and 1702–1705 (immunized with MPER656 peptide-liposomes alone) is shown in comparison to 2F5 mAb. The normalized binding shown is the ratio between binding responses of sera to the alanine scanning mutant peptides and WT 2F5 epitope peptide SP62. Alanine substitution of residues that resulted in $50% reduction in binding are indicated adjacent to the symbols. Data is representative of at least two measurements on adjacent spots on the same sensor chip. doi:10.1371/journal.pone.0027824.g001 PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27824