Discrete partitioning of HIV-1 Env forms revealed by viral capture

Stieh et al. Retrovirology (2015) 12:81 DOI 10.1186/s12977-015-0207-z Open Access RESEARCH Discrete partitioning of HIV‑1 Env forms revealed by viral capture Daniel J. Stieh1, Deborah F. King2, Katja Klein2, Yoann Aldon2, Paul F. McKay2 and Robin J. Shattock2* Abstract Background: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env. Results: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps. Conclusions: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions. Keywords: HIV-1, Monoclonal antibody, Envelope glycoprotein, Viral heterogeneity Background Successful vaccination to prevent HIV-1 acquisition will likely require the elicitation of neutralizing antibodies (nAb) directed against the functional envelope glycoprotein (Env) [1–3]. However, although natural infection rapidly induces Env specific class switched B cells [4], these lack the required specificity to provide potent and broad viral neutralization. Indeed maturation of autologous nAbs requires months to years of persistent systemic infection [3], where <1 % of individuals develop substantial breadth of heterologous neutralization (elite *Correspondence: 2 Mucosal Infection and Immunity Group, Section of Infectious Diseases, Imperial College London, St Mary’s Campus, London W2 1PG, UK Full list of author information is available at the end of the article neutralizers) [5–7]. Thus the features of Env variants that lead to neutralization breadth remain to be defined. The predominance of antibodies that fail to neutralize HIV-1 in infected individuals is thought to be due in part to the diversity of Env conformations presented to the humoral immune system. Virion Env diversity is generated by multiple mechanisms including: the range of virion incorporated Env structures, the degree and extent of Env glycosylation, the flexible nature of the HIV-1 Env protein, and potential instability on the surface of viral particles [8–10]. HIV-1 Env is expressed in infected cells as a non-functional precursor protein gp160. Subsequent proteolytic cleavage of gp160 by cellular furins into non-covalently attached transmembrane gp41 and external gp120 components is a necessary step in the creation of functional © 2015 Stieh et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Stieh et al. Retrovirology (2015) 12:81 Env [11]. The non-covalent nature of the cleaved gp41/ gp120 protein provides an intrinsic instability that is distinct from heterodimeric Env proteins of other viruses such as influenza, which retains a stabilizing disulfide bond [12]. The instability of the gp41/gp120 heterodimer can lead to gp120 shedding leaving gp41 stumps expressed on the virus or infected cells. Although functional Env complexes are thought to require gp41/ gp120 trimerization, variable gp160 processing results in the expression of uncleaved proteins and non-trimeric arrangements of gp41/gp120 heterodimers on the surface of infected cells [13]. Assembly of these Env structures into budding virions provides further variability, with gp41/gp120 incorporated as monomeric, dimeric and trimeric forms [13–15]. Additional diversity is derived from the range of gp120 glycosylation patterns that can obfuscate the Env spike from humoral responses. The array of gp120 N-linked oligomannose glycans is inefficiently trimmed by Golgi and Endoplasmic Reticulum α-mannosidases [16, 17], and this is further reduced by the steric constraints imposed by trimerization [18]. The plasticity of the Env complex itself leads to variable and transient epitope exposure further confounding the potential elicitation of nAb responses [19]. This is enhanced by the fact that a series of conformational rearrangements occur during receptor triggering and viralcell fusion [20, 21]: the co-receptor (CCR5 or CXCR4) binding site is exposed after binding to the CD4 molecule; and the fusion peptide of gp41, that inserts into the target cell and initiates membrane fusion, is only fully exposed following CD4 and co-receptor binding. The inherent flexibility and high potential energy of the Env complex is likely necessary for correct functioning, but this complicates the development of effective mimic immunogens for use in vaccination regimes. Nonetheless, a growing number of broadly neutralizing monoclonal antibodies (mAbs) have been isolated from a few elite controllers that map to sub-regions on the Env spike [22– 24]. These include PG9, PG16, VRC01 and VRC03 mAbs that are understood to recognize gp120 in the context of functional trimers, and display very broad breadth of neutralization in comparison to the previously identified CD4 binding site (CD4bs) b12 mAb and glycan specific 2G12 mAb [25, 26]. Additional very potent broadly nAbs (bnAbs) targeting gp120 have recently been described [22, 27]. In rare instances nAbs have also been isolated from infected s (...truncated)