Induction of MPER Antibodies in Rhesus Figure 8. The JRFL gp140CF primed and MPER656 liposomes boosted rhesus macaque serum IgG bind scaffold proteins containing engrafted MPER in the 2F5 bound conformation. A–C: SPR sensogram of 2F5, 11F10 and 13H11 mAbs at the indicated concentrations binding to ES2 (A), ES4 (B), and ES5 (C) scaffolds respectively are shown. D–F: The post MPER656 liposome immune serum IgG of rhesus macaques (150 mg/ ml) binding to ES2 (D), EJ(E), ES5 (F) are shown. For the sake of clarity, the scaffold binding responses of pre-MPER656 liposome serum IgG of only one animal (# 41546) is shown. The responses from other animal serum IgG were similar. Data is representative of at least two measurements. doi:10.1371/journal.pone.0027824.g008 protein and showed that such antibodies are also capable of binding to 2F5-epitope that is pre-configured in the 2F5 antibody bound state. Thus both immunization strategies, that of Ofek et al. (30) and the present study, have elicited antibodies that recognize the putative fusion-intermediate gp41 conformation. This is underscored by the fact the epitope scaffold induced antibody 11F10, which recognizes the 2F5 bound conformation of gp41, can also recognize the putative gp41 fusion-intermediate confor- mation as presented in the constrained gp41-inter protein. It is notable in the studies of Ofek et al [27] and Guenaga and colleagues [33] that 2F5 scaffolds that were more flexible were better immunogens than those that were rigid. Thus immunogens that allow antigen recognition with induced fit may allow better adaptability to the available na¨ıve B cell repertoire. Our studies using a heterologous prime boost and with MPER presented on liposomes provide an alternative strategy to inducing antibodies with specificity for the 2F5 core epitope, and in all likelihood allowed induced fit binding of 2F5 core epitope-specific antibodies. Table 1. Neutralization data of sera from JRFL gp140CF- MPER656 liposomes immunized rhesus macaques. Animal # ID50 for BG1168.1 ID50 for SF162.LS 03525 41546 51902 51834 Positive control 2F5 mAb ,20 ,20 ,20 ,20 ,20 ,20 ,20 22 Importantly, the antibodies induced by our heterologous prime and boost preferentially bound gp41 fusion- intermediate constructs versus the recombinant gp41 MN. Thus, we have demonstrated that in addition to 2F5 stabilized structures engrafted on acceptor proteins, MPER liposomes when used as a boost can induce gp41-inter protein specific antibodies. It is tempting to speculate that the MPER presentation in JRFL gp140CF is capable of priming for 664DKW responses but this response remains subdominant in the presence of other immuno- dominant gp140 epitopes. We have shown that JRFL gp140CF primarily induces antibody responses that target the recombinant gp41 MN and not the GCN4-gp41 inter protein (Figure S2). We postulate that B cells to the MPER 664DKW were primed by JRFL gp140 and then were expanded when boosted with MPER peptide-liposome complexes lacking other immunodominant epitopes. Although the 2F5 epitope portion of MPER sequence of the priming Env gp140 does not differ (Figure S6) from that used in the MPER liposomes (boost immunogen), the gp140 MPER likely presents a different structure than those on the MPER peptide liposomes. This is evident in the differences in binding affinities of 2F5 or 4E10 to JRFL gp140CF and MPER liposomes [18,19] and in the nature of the induced antibodies (Figure 7 and S2). The heterologous prime/boost strategy with the two MPER structures have thus likely led to the focusing of the antibody responses to the 2F5 core residues and thus resulted in the induction of gp41 inter specific antibodies. Additionally, incorporation of TLR 4 and 7/8 agonists in the MPER peptide- liposome complexes also may have played a role in enhancing immune responses in the boosting stage. In this regard, we have previously reported the partial control of non-neutralizing gp41 MPER antibodies by tolerance controls [17], and others have demonstrated that combinations of TLR agonists including oCpGs and R848 have broken peripheral tolerance and induced autoreactive antibodies [47,48,49,50]. Thus, strong adjuvants and 2.4 mg/ml 3.6 mg/ml Sera from immunized guinea pigs did not neutralize (data not shown). doi:10.1371/journal.pone.0027824.t001 PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27824