Induction of Antibodies in Rhesus Macaques That Recognize a Fusion-Intermediate Conformation of HIV-1 gp41 S. Moses Dennison1, Laura L. Sutherland1, Frederick H. Jaeger1, Kara M. Anasti1, Robert Parks1, Shelley Stewart1, Cindy Bowman1, Shi-Mao Xia1, Ruijun Zhang 1 , Xiaoying Shen1, Richard M. Scearce1, Gilad Ofek4, Yongping Yang4, Peter D. Kwong4, Sampa Santra2, Hua-Xin Liao1, Georgia Tomaras1, Norman L. Letvin2, Bing Chen3, S. Munir Alam1*, Barton F. Haynes1* 1 Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina, United States of America, 2 Department of Medicine, Beth Israel Deaconess Medical Center, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Division of Molecular Medicine, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America Abstract A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide- liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope. Citation: Dennison SM, Sutherland LL, Jaeger FH, Anasti KM, Parks R, et al. (2011) Induction of Antibodies in Rhesus Macaques That Recognize a Fusion- Intermediate Conformation of HIV-1 gp41. PLoS ONE 6(11): e27824. doi:10.1371/journal.pone.0027824 Editor: Paul A. Goepfert, University of Alabama, United States of America Received August 26, 2011; Accepted October 26, 2011; Published November 30, 2011 Copyright: 2011 Dennison et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was conducted as part of the Collaboration for AIDS Vaccine Discovery (CAVD) with support from the Bill & Melinda Gates Foundation to BFH (38643) and from the Center for HIV/AIDS Vaccine Immunology (CHAVI) AI067854 from the NIH, NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alam0004@mc.duke.edu (SMA); hayne002@mc.duke.edu (BFH) Introduction The membrane proximal external region (MPER) of HIV-1 gp41 contains the epitopes of broadly neutralizing antibodies 2F5 and 4E10 [1,2,3,4] that are important targets for HIV-1 vaccine design. The MPER of gp41 is a highly conserved region, rich in aromatic residues, and its role in HIV-1 fusion is evident from studies showing that mutation of tryptophan residues in the MPER inhibits cell fusion and viral infectivity [5,6]. Passively adminis- tered neutralizing antibodies 2F5 and 4E10 can protect against vaginal SHIV transmission [7] indicating that if induced in high titers, such broadly neutralizing antibodies could be effective against HIV-1 infection. However, MPER-specific broadly neutralizing antibodies are rarely made in HIV-1 infection [8,9] or following HIV-1 envelope protein (Env) vaccination [10,11,12,13]. Structural constraints that include transient expo- sure of neutralizing epitopes [14,15], as well as immunological tolerance mechanisms [16] are explanations for inability to routinely induce 2F5 or 4E10-like antibody responses. Thus, there are two barriers to induction of MPER broad neutralizing antibodies that must be overcome; the transient exposure and poor immunogenicity of subdominant MPER neutralizing epitopes, and tolerance control of the B cells capable of responding to the MPER neutralizing epitopes [16,17]. The high affinity binding of 2F5 and 4E10 mAbs to the membrane-displayed MPER followed a two-step encounter- docking model that was distinct from the binding pattern observed in MPER antibody interaction with gp41 epitopes in the absence of lipids [18,19]. These and other data suggest that MPER residues are orientated or presented differently on a lipid bi-layer compared to free peptides [15,19,20,21]. The lack of binding of a non-neutralizing MPER mAb 13H11, the binding site of which overlaps that of 2F5, to MPER peptide-liposomes [18], further highlights the differences in configuration of the MPER residues in peptide-lipid complexes when compared to MPER peptide in solution. In contrast, gp41 peptides exist in multiple conformations - unstructured, a-helical, or b-turn [22,23,24] and MPER peptides alone as immunogens generally are ineffective for the induction of neutralizing antibodies (reviewed in [25]). MPER peptides in micelles or in liposomes with membrane anchor tags are likely to be less flexible and more likely to adopt a relatively ordered conformation [19,20,21,26]. Ofek et al., recently showed that PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27824