Induction of MPER Antibodies in Rhesus Figure 7. Comparison of JRFL gp140CF primed and MPER656 liposomes boosted rhesus macaque serum IgG binding to different conformational states of gp41. A–D: SPR sensogram of rhesus macaques serum IgG (100 mg/ml) binding to GCN4-gp41 inter (solid lines), gp41- inter (dotted lines) and recombinant gp41 (broken lines). The IgGs purified from Week 56 (for animals 03525 and 51902), Week 52 (animal 41546) and Week 52 (animal 51834) sera were used. Data is representative of two measurements. doi:10.1371/journal.pone.0027824.g007 VH knock-in mice [16], and in 2F5 VH+VL knock-in mice (Verkoczy L and Haynes B, unpublished). Several earlier studies have used specialized structures to present the MPER sequences in the immunogens. Arnold et al had used recombinant human rhinoviruses displaying ELDKWA epitope flanked by residues promoting b- turn [35], Zhang et al expressed chimeric virus like particles presenting a bovine papillomavirus L1-HIV-1 gp41 fusion protein [36] and Liang et al had grafted ELDKWA into variable loops in HIV-1 gp120 [37]. These and other studies have reported induction of antibodies that target the 2F5-nominal epitope but with minimal or no neutralization [24,35,36,37,38,39,40]. Although in these studies, induced antibody responses were targeted to the ELDKWA sequence, fine specificity mapping data are not available for determination if the antibody responses were restricted to the core tripeptide 664DKW. Similarly, in studies with immunogens with 4E10 epitope, the lack of fine specificity data does not allow assessment of the epitopes induced by the immunogens [13,41]. More recently, however, MPER directed neutralizing antibodies were reported to be elicited using immunogens consisting of gp41 six helix bundle and an exposed MPER tail with double mutation [42] and a chimeric construct in which the HIV gp120 was replaced with HA1 subunit of influenza virus [43]. That weak MPER neutralizing activity can sporadically be induced and detected is not surprising, since a subset of chronically HIV-1 infected subjects have been reported to make neutralizing antibodies that bind within the MPER [9,44,45]. However, high and sustained levels of MPER antibodies are not induced in the setting of vaccination and this may relate to immunoregulatory control of poly reactive MPER antibodies with more potent neutralizing activity such as 2F5 and 4E10 [16,46]. Guenaga et al. [33] and Ofek and colleagues [27] recently demonstrated that a heterologous scaffold prime-boosting strategy induced antibody responses to the engrafted 2F5 epitopes, and that these antibodies bound to the MPER peptide that existed in the 2F5 bound conformation. These latter studies did not determine reactivity with gp41 fusion-intermediate forms, nor did these scaffold-induced antibodies neutralize HIV-1. However, like the MPER liposomes induced antibodies described in this study, one antibody, 11F10, isolated from the above 2F5 scaffold murine immunization study [27] also bound preferentially to the gp41-inter conformation and not to the recombinant gp41 MN (Figure S3). In this study we have described the induction of antibodies that preferentially bind to a gp41 fusion- intermediate PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27824