Induction of MPER Antibodies in Rhesus Figure 3. Comparison of MPER specific immunogenicity of different immunogens in rhesus macaques. The immunization scheme is shown at the top. A–B: MPER specific binding responses of sera from immunized rhesus macaques at different time points are shown for (A) JRFL gp140CF (ASO1B adjuvant) and (B) MPER peptide liposome immunogens (in emulsigen+oCpG). C–E: The fine specificities of MPER specific responses elicited by MPER liposomes in rhesus macaques are shown for post immunizations 2–5 sera. F: The alanine substituted peptides used in epitope mapping of MPER responses. The dotted ellipses in C, D and E highlight the predominance of 664D specific MPER responses. Data is representative of at least two measurements on adjacent spots on the same sensor chip. doi:10.1371/journal.pone.0027824.g003 inconsistent involvement of 665K of the 2F5 core tripeptide 664DKW (Figure 3C–E). Thus, antibody responses with the desired amino acid targeting (664DKW) were not achieved in rhesus macaques immunized with MPER peptide-liposomes or JRFL gp140CF oligomer alone. Rhesus macaque antibody responses to heterologous prime-boost immunization Since we observed that heterologous prime-boost immuniza- tions in guinea pigs were more effective in inducing 664DKW - specific antibody responses, we next tested whether the JRFL gp140CF prime-MPER peptide-liposome boost regimen would also focus the induced antibody responses on the gp41 664DKW neutralizing epitope in rhesus macaques. The Env prime, MPER- liposome boost immunization regimen used in rhesus macaques is shown in Figure 4A (same animals in 3A were boosted). The MPER specific responses in sera from four immunized animals at different points are shown in Figures 4B–C. MPER specific responses began to appear in sera collected 3 weeks after first MPER peptide-liposome complex boost (week 51). Each subsequent boosting enhanced the level of 2F5 peptide binding responses, with two animals having about 3-fold higher binding responses than the remaining two (Figure 4B–C). The responses declined during the interval between the 2nd and 3rd boost but then increased again following the 3rd MPER peptide-liposome complex boost (Figure 4B–C). Serum antibody binding dissoci- ation rates, a measure of binding avidity, was stable over time (Figure 4D). Fine specificity mapping of induced antibody demonstrated that the MPER-specific responses elicited after the first boost of MPER liposomes were targeted to one or two core 664DKW residues and as well targeted additional MPER residues (L669, W670), outside the 2F5 core but within the MPER hinge region (Figure 5). However, following the second and third MPER peptide-liposome complex boosts, rhesus monkey serum antibody responses to 664DKW residues were consistently induced (Figures 5A–D). In 3 out of 4 animals (03525, 41546 and 51902), sera collected one week after the third boost (week 56) showed MPER specific responses in which all three 664DKW residues were recognized by serum antibodies (Figures 5A,B and D). In the fourth animal (51834), the specificity for all three DKW residues was also observed in week 55 bleed but with weaker and transient involvement with W666 in week 56 (Figure 5C). Thus, these results indicated that using a heterologous prime-boost regimen of JRFL gp140CF/ MPER peptide-liposome complexes, MPER-specific antibody responses in rhesus macaques could be focused to MPER 664DKW amino acids. PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27824