Induction of MPER Antibodies in Rhesus Figure 5. Mapping of critical residues involved in MPER specific Rhesus macaques serum responses. Normalized binding responses of immune sera from rhesus macaques primed with JRFL gp140CF and boosted with MPER656 liposomes. Panels A–D are for animals 03525, 41546, 51384 and 51902. The dotted circle highlights the mapping of MPER specific responses to D664KW residues as did 2F5 mAb. Data is representative of at least two measurements on adjacent spots on the same sensor chip. doi:10.1371/journal.pone.0027824.g005 that had the epitope in a 2F5-bound conformation possessed nanomolar affinity for 2F5 mAb with varying degrees of binding entropy [27]. Crystal structure analysis of one of the scaffold (ES2) which had the highest affinity towards 2F5 and the most entropically favorable interaction with 2F5 showed that the epitope conformation mimicked that of the 2F5-bound conformation [27]. Thus, we examined the binding of pre- (week 48) and post- MPER656 liposome (weeks 52–56) immune serum IgG of rhesus macaques to 2F5-epitope scaffolds ES2, ES4 and ES5. As previously reported [27], 2F5 mAb and 11F10, a mouse monoclonal antibody elicited in a ES5–ES1 prime:boost immunization regimen bound strongly to the ES proteins (8A–C). The non-neutralizing 13H11, which recognizes the 2F5 epitope core (664DKW) and L669 [15] in a helical conformation [28] did not bind the 2F5-epitope scaffolds (Figure 8A–C). Interestingly, the post-MPER656 liposome immune serum IgG of rhesus macaques bound to 2F5-epitope scaffolds ES2, ES4 and ES5 (Figure 8D–F). As also observed with GCN4-gp41- inter binding, the post JRFL gp140 induced antibodies (week 48) did not bind to any of the scaffolds (Figure 8D–F). To determine if the ES2 scaffold shares antigenicity with the gp41 inter proteins, we determined the ability of the 11F10 mAb to bind to gp41-inter proteins. We found that 11F10 bound to gp41 inter proteins (Kd = 7.9 and 7.8 nM to GCN4-gp41 inter and 92Ug gp41-inter respectively), but not to the gp41 MN protein (Figure S3). 11F10 mAb, therefore, selectively binds to the putative fusion-intermediate conformation of gp41. However, unlike 2F5 mAb 11F10 did not bind anionic lipids (Figure S4) nor neutralized HIV-1 [33]. Thus, these data indicate that the 664DKW specific antibodies elicited in rhesus macaques can not only recognize fusion-intermediate protein but can also bind to 2F5-bound conformation of gp41 MPER. Neutralization capacity of rhesus monkey serum antibodies that bind to gp41 fusion-intermediate protein Having elicited 664DKW-specific antibodies that recognize the gp41 fusion-intermediate conformation and 2F5-bound confor- mation in rhesus macaques, we asked whether these responses would neutralize HIV-1. In TZM/bl pseudovirus assays (Table-1) PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27824