Tolbert et al. BMC Biology (2020) 18:91 https://doi.org/10.1186/s12915-020-00819-y RESEARCH ARTICLE Open Access Defining rules governing recognition and Fc-mediated effector functions to the HIV-1 co-receptor binding site William D. Tolbert1,2†, Rebekah Sherburn1,2†, Neelakshi Gohain2†, Shilei Ding3,4, Robin Flinko2, Chiara Orlandi2, Krishanu Ray1, Andrés Finzi3,4,5, George K. Lewis2 and Marzena Pazgier1,2* Abstract Background: The binding of HIV-1 Envelope glycoproteins (Env) to host receptor CD4 exposes vulnerable conserved epitopes within the co-receptor binding site (CoRBS) which are required for the engagement of either CCR5 or CXCR4 co-receptor to allow HIV-1 entry. Antibodies against this region have been implicated in the protection against HIV acquisition in non-human primate (NHP) challenge studies and found to act synergistically with antibodies of other specificities to deliver effective Fc-mediated effector function against HIV-1-infected cells. Here, we describe the structure and function of N12-i2, an antibody isolated from an HIV-1-infected individual, and show how the unique structural features of this antibody allow for its effective Env recognition and Fc-mediated effector function. Results: N12-i2 binds within the CoRBS utilizing two adjacent sulfo-tyrosines (TYS) for binding, one of which binds to a previously unknown TYS binding pocket formed by gp120 residues of high sequence conservation among HIV-1 strains. Structural alignment with gp120 in complex with the co-receptor CCR5 indicates that the new pocket corresponds to TYS at position 15 of CCR5. In addition, structure-function analysis of N12-i2 and other CoRBS-specific antibodies indicates a link between modes of antibody binding within the CoRBS and Fc-mediated effector activities. The efficiency of antibody-dependent cellular cytotoxicity (ADCC) correlated with both the level of antibody binding and the mode of antibody attachment to the epitope region, specifically with the way the Fc region was oriented relative to the target cell surface. Antibodies with poor Fc access mediated the poorest ADCC whereas those with their Fc region readily accessible for interaction with effector cells mediated the most potent ADCC. Conclusion: Our data identify a previously unknown binding site for TYS within the assembled CoRBS of the HIV-1 virus. In addition, our combined structural-modeling-functional analyses provide new insights into mechanisms of Fc- effector function of antibodies against HIV-1, in particular, how antibody binding to Env antigen affects the efficiency of ADCC response. Keywords: HIV-1, Env, Co-receptor binding site, ADCC, N12-i2 * Correspondence: [email protected] †William D. Tolbert, Rebekah Sherburn and Neelakshi Gohain contributed equally to this work. 1Infectious Disease Division, Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4712, USA 2Division of Vaccine Research of Institute of Human Virology, University of Maryland School of Medicine, Baltimore, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tolbert et al. BMC Biology (2020) 18:91 Page 2 of 20 Background Historically, such antibodies were classified solely based The human immunodeficiency virus (HIV-1) contains on their direct neutralizing activities; not much was only one viral glycoprotein on its surface; the envelope known about their involvement in Fc-mediated processes. (Env) spike, which is responsible for both target cell rec- Capable of direct neutralization of mostly Tier 1 viruses, ognition and virus-host fusion. The Env spike consists of in the absence of complement or effector cells, CoRBS a non-covalent trimeric assembly of exterior gp120 and Abs were viewed as moderately to weakly neutralizing and transmembrane gp41 glycoproteins [1, 2]. During viral overall not capable of neutralizing most circulating iso- entry, the recognition step is mediated by the specific lates [9, 29, 30]. Furthermore, the neutralization potency interaction of the exterior gp120 subunit with the pri- in this class was shown to directly correlate with unusual mary receptor at the target cell surface, CD4 [3]. CD4 antibody structural features with the most effective neu- binding induces conformational changes in the trimer, tralizers having long, tyrosine-O-sulfated CDR H3s [31– leading to the formation of the gp120 bridging sheet 35]. It was shown that CoRBS Abs containing tyrosines which, together with the stem of the gp120 third variable modified by post-translational O-sulfation were able to region (V3), constitutes the Env interaction site for co- mimic the interaction mediated by the N-terminus of receptor binding (CoRBS), allowing either CCR5 or CCR5, which contains up to four O-sulfo-tyrosines, with a CXCR4 to bind [4, 5]. Together, CD4-triggering and en- minimum of two required for HIV-1 cell entry [34, 36]. gagement of a co-receptor enable fusion of the viral and We recently performed a systematic analysis of a panel host cell membranes. The transitional structures arising of 33 CD4i monoclonal antibodies (mAbs) directed at the within the Env spike post-CD4 attachment in the HIV-1 CoRBS, Cluster C region, and defined their fine epitope entry process are referred to as “CD4-induced” (CD4i) specificities, neutralization, and ADCC potencies [15, 19]. and constitute effective targets for humoral immune re- We detected, consistent with previous reports [9, 29, 30, sponses [6–10]. These CD4i targets include highly con- 32–35], a substantial neutralization diversity among anti- served epitopes that are located within/proximal to the bodies directed at Cluster C epitopes, with the most po- assembled CoRBS [11–14] and epitopes elsewhere in the tent being those that bind epitopes overlapping the protein, including the C1/C2 region of gp120 [6–8, 15– classical 17b epitope and having tyrosine-O-sulfated CDR 18]. The CoRBS and C1C2 epitopes are commonly re- H3s. Most interestingly, our studies were the first to sys- ferred to as Cluster C and Cluster A epitope regions, re- tematically rank the breadth and potency of CoRBS epi- spectively [15, 19]. CD4i epitope targets, including the topes as functional ADCC targets. We showed that most Cluster C and A regions, are not fully exposed on the mAbs specific for these epitopes mediated significant free Env spike, and anti-Env antibodies specific for these ADCC against target cells coated with AT-2 inactivated regions show enhanced affinity for the gp120-CD4 com- viral particles [15]. Interestingly, the CoRBS antibodies plex as compared to un-triggered gp120 [20]. The struc- were also recently found to act synergistically with Cluster ture of gp120 in complex with both CD4 and CCR5 has A antibodies to promote FcγRIIIa engagement and to me- recently been solved by cryo-electron microscopy and diate ADCC of CD4+ HIV-infected cells and HIV-infected revealed that CCR5 most likely acts to stabilize the cells pre-treated with small CD4 mimetics [19, 37, 38]. gp120 in CD4-bound conformation and to moor the Here, we selected the mAb N12-i2, a representative anti- complex close to the cell surface [21]. body of the Cluster C panel [15], and described the molecu- Several lines of evidence point towards the involvement lar details of its interaction with the Env antigen. N12-i2, of the CD4i antibody class in protection against HIV-1 ac- similar to other CoRBS-specific antibodies, binds within the quisition or/and post-infection control during natural in- assembled co-receptor binding site but utilizes a unique fection and in vaccine settings [17, 22–24]. The CD4i binding mode that involves two sulfoTYSs, one of which oc- Cluster A antibodies were implicated in the protective ef- cupies a newly defined TYS binding pocket. Furthermore, in fect of the RV144 vaccine regimen in humans [25, 26]and order to better understand the mechanisms governing Fc- a mix of Cluster A and CoRBS antibodies induced by the effector mechanism to the CoRBS epitopes, we examined if covalently constrained gp120-CD4 complex vaccine (full- the fine epitope specificity and mode of binding of N12-i2 length single chain (FLSC) vaccine [27]) afforded heterol- and other CoRBS-specific antibodies contribute to the ef- ogous protection against SHIV162p3 and SIVmac251 in fectiveness of Env engagement and Fc-mediated effector ac- non-human primate (NHP) challenge studies [23, 28]. tivities in the elimination of CEM-NKR cells sensitized with Interestingly, Cluster A antibodies impacted the virus HIV-1 gp120 BaL or infected with the ADA virus. solely through Fc-mediated effector mechanisms, without direct neutralizing activity and were capable of cytolytic Results activity through antibody-dependent cellular cytotoxicity N12-i2 targets the co-receptor binding site of HIV-1 Env (ADCC) [15]. In contrast, less is known about the exact As initially assessed [15], N12-i2 recognizes the transi- mechanisms of the protective action of CoRBS antibodies.