In-Solution Virus Capture Assay Helps Deconstruct Heterogeneous Antibody Recognition of Human Immunodeficiency Virus Type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (MAbs) is challenging. Virus capture assays (VCAs) involving immobilized MAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing MAbs, including 2G12, 4E10, 2F5, Z13e1, and D5, will capture virion particles completely devoid of Env. We modified the VCA such that MAbs and virions are incubated in solution, and unbound MAbs are removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by MAbs to virions and allowed determination of apparent affinity constants in solution. Three important qualitative observations were further revealed. First, neutralizing MAbs 2F5, 4E10, and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype and that Env from HIV-1 JR-FL is more homogeneously trimeric than that from HIV-1 JR-CSF . Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular "bridge" between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.Eliciting neutralizing antibody (Ab) against human immunodeficiency virus type 1 (HIV-1) is a crucial but exceedingly difficult challenge in HIV-1 vaccine design (10, 32). The HIV-1 envelope glycoprotein (Env) is the specific target of all HIV-1 neutralizing Abs that have been identified to date (87,98). Env is produced as a gp160 precursor molecule that is cleaved by cellular proteases into a surface subunit, gp120, and a transmembrane subunit, gp41, which in the functional state of Env are assembled as noncovalent trimers of gp120-gp41 heterodimers (45, 91). The Env trimer engages host cell CD4 and coreceptor (CCR5 or CXCR4) through interaction with gp120, and this elicits conformational changes in gp41 that facilitate the subsequent fusion of virus and host cell membranes (29). However, native Env trimers coexist with distinct, nonfunctional forms of Env (34, 51, 65). These nonfunctional forms, including nontrimeric and aberrant disulfide-linked forms of Env, gp41 stumps from which gp120 has been shed, and uncleaved gp160, appear to be highly immunogenic but tend to elicit non-neutralizing antibodies (51,60,94).During the acute phase of natural infection, non-neutralizing Abs are commonly elicited, particularly to gp41 (83). Neutralizing Ab responses develop over time, but these tend to be is...

Antigenic Properties of the HIV Envelope on Virions in Solution

“…For example, neutralizing anti-MPER MAbs capture virions only weakly (47), whereas poorly neutralizing antibodies capture particles relatively well (40). The latter effects have been attributed to a preponderance of misfolded trimers on virion surfaces, potentially introduced in part by the capture method, and/or to avidity effects that can produce artificially strong virion binding to immobilized antibodies (40,45). A means to directly analyze antibody-virion interactions with all reactants in solution could present a more unalloyed picture of surface epitope exposure under key in vivo and/or in vitro conditions.…”

“…To date, analyses of envelope antigenicity on HIV particles have relied heavily on various types of assays in which epitope exposure is measured as a function of virion capture by immobilized MAbs. The captured virions are typically detected by indirect means such as infectivity assays (40,(42)(43)(44)(45)(46). This methodology has produced discordant findings regarding the neutralizing versus capture activities of anti-envelope MAbs (43).…”

Nanoparticle-based flow virometry for the analysis of individual virions

“…Up to now, the presence of these proteins has been studied in viral preparations either with ELISA or with flow cytometry of multiple viruses bound to microbeads (9,12,(22)(23)(24)(25)(26)(27). These studies analyzed viral particles in bulk.…”

Dense Array of Spikes on HIV-1 Virion Particles

“…The augmentation of Env trimers on cells and progeny hVLPs thus provides opportunities for vaccine design that includes native Env in a membrane environment. spikes on cognate virions (30). Our attempts to increase Env content using DNA transfection, including env codon optimization, use of a constitutive cytomegalovirus (CMV) promoter, optimized leader sequence, and truncation of the CTT did not significantly increase the number of mature Env trimers on virions but did produce an excess of immature or misfolded Env debris (30).…”

“…spikes on cognate virions (30). Our attempts to increase Env content using DNA transfection, including env codon optimization, use of a constitutive cytomegalovirus (CMV) promoter, optimized leader sequence, and truncation of the CTT did not significantly increase the number of mature Env trimers on virions but did produce an excess of immature or misfolded Env debris (30). We considered that impediments to a dense display of spikes on the membrane may be intrinsic to the producer cell.…”