Induction of MPER Antibodies in Rhesus Figure 4. MPER specific binding responses of Rhesus macaques sera in gp140 prime-MPER liposome boost regimen. A: The immunization scheme involving JRFL gp140CF prime (ASO1B adjuvant) and MPER656/MPL-A/R848 (emulsigen+oCpG) liposome boost regimen is shown. The arrows indicate the weeks at which immunization was done. B: ELISA end point titer of the 2F5 epitope peptide specific responses in rhesus macaques sera at different time points. C: The 2F5 epitope peptide specific binding of monkeys sera determined by SPR is shown post vaccination at different time points. D: Representative data of SPR sensogram of rhesus macaque #41546 sera at different time points binding to 2F5 epitope peptide as compared to 2F5 mAb (10 mg/ml) is shown. The estimated dissociation rates (kd) are indicated for samples at week 51 and 56 as well as for the control 2F5 mAb. Data is representative of at least two measurements on adjacent spots on the same sensor chip. doi:10.1371/journal.pone.0027824.g004 Induction of antibodies to gp41 fusion-intermediate protein by Env gp140 prime, MPER liposome boost regimen While induction of pre-hairpin intermediate antibodies is difficult, induction of post-fusion six-helix bundle antibodies is common [17,29,30,31]. The neutralizing MPER antibodies 2F5 and 4E10 target the fully extended fusion intermediate state of HIV-1 gp41 [14,32] whereas non-neutralizing MPER antibody 13H11 recognizes a helical MPER structure that is consistent with HIV-1 gp41 in a post-fusion six-helix bundle conformation [28]. To fully understand the gp41 conformation targeted by the induced antibodies, we tested three different recombinant gp41 proteins; the gp41MN protein, and two gp41-inter constructs- gp41-inter and GCN4 gp41-inter [14,32]. While both trimeric gp41 inter proteins present the MPER in its putative fusion intermediate conformation, gp41-inter construct has an additional six-helix bundle attached at its N-terminus end [14,32]. Thus antibodies that exclusively recognize the fusion intermediate conformation of gp41 will bind to both the gp41 inter proteins, while those that do not target the fusion intermediate will bind to gp41 and/or gp41-inter but not GCN4-inter [32]. JRFL gp140CF immunized macaques sera (week 14, Figure S2), which gave no MPER peptide binding, bound to both gp41 MN and gp41-inter but not to GCN4 gp41-inter. The gp41 specific antibodies induced by JRFL gp140CF, therefore, are unable to recognize the fusion intermediate conformation of gp41. Similarly, the week 48 sera (following priming with JRFL gp140CF protein), which showed no detectable MPER specific responses by SPR, also gave no binding to GCN4-gp41 inter (Figure 6B–E). Remarkably the post- MPER656 liposome boost sera (Weeks 52–56) that gave DKW- specific responses bound strongly to GCN4-gp41 inter (Figure 6B– E). Since GCN4-gp41 inter is recognized by 2F5 and not by 13H11 (Figure 6A), the GCN4-gp41 inter binding of MPER specific responses in these rhesus macaques sera indicated that the induced DKW-specific antibodies were capable of recognizing the fusion-intermediate state of gp41. Furthermore, as shown in Figure 7, the purified IgG binding responses gave higher binding to both gp41-inter and GCN4-inter while the binding to gp41 MN was much lower. These results indicated that MPER peptide- liposomes boost can focus the antibody responses to the 2F5 core DKW residues and induce antibodies in rhesus macaques that preferentially bound the MPER conformation in the fusion- intermediate protein. Thus, the JRFL gp140CF/MPER peptide- liposome complex regimen has solved one component of the problem for induction of neutralizing anti-gp41 MPER antibodies, i.e., induction of antibodies with specificity for a fusion- intermediate conformation of gp41 MPER. JRFL gp140CF prime, MPER liposome boost regimen induced antibodies bind to 2F5-epitope scaffold proteins We next asked whether the JRFL gp140CF prime:MPER656 liposome boost regimen induced antibodies recognize the 2F5 epitope in the 2F5-bound conformational state. Ofek et al recently demonstrated that computationally designed 2F5-epitope scaffolds PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27824