Induction of MPER Antibodies in Rhesus Figure 6. The MPER specific serum responses in rhesus macaques recognize the mimic of fusion-intermediate gp41 structure. A: Comparison of binding of 2F5 and 13H11 Fabs to GCN4-gp41-inter, a mimic of pre-hairpin intermediate structure of gp41. B–E: SPR sensogram of rhesus macaques sera (1:50 diluted) binding to GCN4-gp41-inter. Each panel in B thru E compares the binding of sera obtained prior to MPER656 liposomes boost (week 48) and post-MPER656 liposomes boost (weeks 52–56). Data is representative of two measurements. doi:10.1371/journal.pone.0027824.g006 no significant neutralization was detected in rhesus macaque sera taken at week 56 when the 664DKW core antibody responses were observed. The lipid reactivity of 2F5 and 4E10 through the hydrophobic residues in the CDR H3 loop is critical for HIV-1 neutralization but not for antigen binding [29]. Thus, we asked whether the GCN4-gp41 inter binding 664DKW-specific antibodies elicited in rhesus macaques had any lipid reactivity. Purified IgG from rhesus macaque sera at weeks 0, 48, 52, and 55 was tested for reactivity to liposomes containing cardiolipin, or phosphatidylserine (PS) that do bind to mAb 2F5 [34]. There was no specific binding of rhesus macaque IgG purified from either the pre- or the post-vaccination sera to either cardiolipin (Figure S5) or PS (not shown). Taken together these results suggested that the induced DKW-specific rhesus macaque antibodies lacked the ability to bind lipids, and were therefore, unable to neutralize HIV-1. Discussion In this study, we report that the membrane bound form of gp41 MPER peptides can present epitopes in a conformation that induce antibodies that not only target the core 664DKW epitope of the neutralizing antibody 2F5, but also recognize a fusion- intermediate construct of HIV-1 gp41 MPER as well as the 2F5 bound MPER conformation. The induction of antibodies with specificity for 2F5 tripeptide core residues was acquired only after priming with an oligomeric gp140, followed by multiple boosts with MPER liposomes, implying that successive boosting was critical for the expansion and maturation of B cells with receptors that target the core 664DKW MPER epitope. Although the induced responses were not neutralizing, the success of this immunization strategy to induce a putative fusion intermediate/ 2F5 bound conformation recognizing antibodies provides a solution to one barrier to inducing gp41 neutralizing antibodies. One explanation for the failure of earlier vaccines is that the immunogens used were not configured in the conformation needed to elicit a gp41 fusion-intermediate antibody response. Our current regimen overcomes this problem. However, during HIV-1-induced CD4 T cell fusion, the MPER is only exposed transiently [14]. In order to capture such a transient epitope we have hypothesized that elicited antibodies should be capable of concentrating on virion surfaces by weak interactions with viral membrane lipids [29]. We have also hypothesized that such antibodies might not be easily made since lipid or protein autoreactivity of MPER specific antibodies may evoke tolerance controls [34]. Indeed, this has been shown to be the case in 2F5 PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27824