An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions

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...

Section: Discussionmentioning

confidence: 99%

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

Leaman

Kinkead

Zwick

2010

“…All attempts to elicit 2G12-like antibodies with the desired specificity and neutralization activity have failed to date (22,29,44), conceivably due to difficulties in generating adequate mimicry of the glycan cluster and tolerance mechanisms or, very likely, the inability to induce domain exchange (1). Unraveling the mechanism of domain exchange and how this conformation might have evolved is highly desirable to direct future HIV-1 vaccine design to elicit 2G12-like antibodies.…”

mentioning

confidence: 99%

Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

Huber

Le²,

Doores

et al. 2010

2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V H ) with the V H of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V H and C H 1 and an extensive network of hydrophobic interactions in the V H /V H interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A H14 and E H75 , are the most crucial for domain exchange, together with I H19 at the V H /V H interface and P H113 in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domainexchanged antibody response in vivo may be more readily achieved than considered to date.

“…While several immunogens displaying multivalent high-mannose glycans have been developed that support 2G12 binding, their affinity is typically much lower than that of 2G12 for gp120, and thus far none have elicited antibodies that cross-react with glycans present on HIV-1 Env, let alone neutralize virus (1,2,11,16,34). However, genetic modification of the S. cerevisiae N-linked glycan processing pathway, such that endogenous glycoproteins express exclusively high-mannose glycans like those recognized by 2G12, resulted in a yeast strain (TM yeast) that mimics at least some aspects of the 2G12 epitope.…”

Section: Discussionmentioning

confidence: 99%

“…In each case, the goal is to present a multivalent array of oligomannose glycans bearing terminal Man␣1,2-Man moieties in an immunogenic context. One approach involves conjugation of oligomannose (Man [4][5][6][7][8][9] carbohydrates, singly or as oligodendrons, to carrier proteins with multiple conjugation sites, such as bovine serum albumin (BSA), cyclic peptides, or viral capsid proteins (1,2,16,17,33,34). An alternative approach employs modification of glycosylation in yeast or mammalian cells to force retention of highmannose glycans on natural proteins (11,18,19,28).…”

mentioning

confidence: 99%

Yeast-Elicited Cross-Reactive Antibodies to HIV Env Glycans Efficiently Neutralize Virions Expressing Exclusively High-Mannose N-Linked Glycans

Agrawal-Gamse

Luallen

Liu

et al. 2011

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus.