2F5 core tripeptide 664DKW residues that are critically involved in mAb 2F5 neutralization. Since we observed weak 2F5 epitope specific responses with JRFL gp140CF alone and only partial focusing on the 2F5 core epitope with MPER peptide liposomes, we next asked whether a heterologous prime-boost regimen using both immunogens would induce antibody responses with specificity restricted to the core tripeptide 664DKW residues. To determine the optimal prime-boost regimen, we used two different strategies. First, we tested a MPER peptide-liposome prime-JRFL gp140CF boost regimen as shown in Figure 2A (same animals in Figure 1B were boosted). In the second strategy, we used a single JRFL gp140CF prime followed by MPER liposome boost (36) as shown in Figure 2D. The MPER peptide-liposome prime-JRFL gp140CF boost regimen resulted in immune responses (Figure 2B) that targeted either 664D or 665K (Figure 2C) and were not different from the results obtained with MPER peptide-liposome alone immunizations. In contrast, as shown in Figures 2D–F, a single prime with JRFL gp140CF followed by MPER peptide- liposomes elicited 2F5 peptide specific responses (Figure 2E) that targeted two of the tripeptide core residues, 664D and 665K, in all three animals (Figure 2F). Thus, the strategy of priming Induction of MPER Antibodies in Rhesus once with JRFL gp140CF and boosting (36) with MPER peptide-liposomes induced antibody specificity that was closer to that of the neutralizing antibody 2F5. In a separate study, the induction of 2F5-like specificity was reproducible and in one animal two rounds of prime-boost immunizations progressively focused the response to the 2F5 core tripeptide 664DKW residues (Figure S9). Non-human Primate (NHP) serum antibody responses to JRFL gp140CF and MPER peptide-liposomes To determine if the strategy of gp140 prime/MPER peptide- liposome complex boost for induction of anti-664DKW antibodies would translate to non-human primates, we next examined the immunogenicity of JRFL gp140CF alone and MPER peptide- liposomes alone in rhesus macaques. Immunization of JRFL gp140CF oligomer alone induced weak (,1:100 titer) MPER- specific responses in rhesus macaques (Figure 3A) although end- point antibody titers against the JRFL gp140CF Env protein were $1/10,000 (Figure S1). In contrast, MPER peptide-liposome complexes alone did induce MPER-specific antibodies (Figure 3B), but the specificity of the MPER liposome-induced responses was restricted to residue 664D, with weak and Figure 2. MPER specific immunogenicity and fine specificity of different prime-boost regimens in guinea pigs. A–C: Immunization scheme (A), 2F5 epitope peptide (SP62 peptide) specific binding serum responses (B) and fine specificity of post immune bleed 7 (C) are shown for MPER656 liposome prime-JRFL gp140CF boost regimen are shown. PB, pre-bleed; P1–P7, post-immune bleeds 1–7. D–F: The JRFL gp140CF prime- MPER656 liposome boost regimen scheme (D), 2F5 epitope peptide specific binding serum responses J and the fine specificity of post immune bleed 4 (F) are shown. Data is representative of at least two measurements on adjacent spots on the same sensor chip. doi:10.1371/journal.pone.0027824.g002 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27824