Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1

Jeffs, Simon A.; S, Goriup; Kebble, Benjamin; Crane, Denis I.; Bolgiano, Barbara; Sattentau, Quentin J.; Jones, Samuel Adeniyi; Holmes, Harvey

doi:10.1016/j.vaccine.2003.08.042

Cited by 48 publications

(59 citation statements)

References 49 publications

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“…The antibodies B18, C4, and CA13 have been reported to recognize linear epitopes within helix 1 of gp120 (62,82), and we confirmed their specificity in a peptide competition ELISA (see Fig. S19 in the supplemental material).…”

Section: Figsupporting

confidence: 78%

“…Such constructs remain elusive despite substantial efforts (96). Soluble oligomeric gp140 ectodomain constructs, which are often thought to mimic the native Env trimer, appear to exhibit substantial heterogeneity from one isolate to another (82), and in many cases, they adopt nonnative conformations, especially within gp41 (M. Guttman et al, unpublished data). At present, full-length gp120s are the most tractable system for comparing structural order among isolates.…”

Section: Discussionmentioning

confidence: 99%

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Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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dThe HIV-1 envelope glycoprotein (Env) mediates viral entry into host cells and is the sole target of neutralizing antibodies. Much of the sequence diversity in the HIV-1 genome is concentrated within Env, particularly within its gp120 surface subunit. While dramatic functional diversity exists among HIV-1 Env isolates-observable even in the context of monomeric gp120 proteins as differences in antigenicity and immunogenicity-we have little understanding of the structural features that distinguish Env isolates and lead to isolate-specific functional differences, as crystal structures of truncated gp120 "core" proteins from diverse isolates reveal a high level of structural conservation. Because gp120 proteins are used as prospective vaccine immunogens, it is critical to understand the structural factors that influence their reactivity with antibodies. Here, we studied four full-length, glycosylated gp120 monomers from diverse HIV-1 isolates by using small-angle X-ray scattering (SAXS) to probe the overall subunit morphology and hydrogen/deuterium-exchange with mass spectrometry (HDX-MS) to characterize the local structural order of each gp120. We observed that while the overall subunit architecture was similar among isolates by SAXS, dramatic isolate-specific differences in the conformational stability of gp120 were evident by HDX-MS. These differences persisted even with the CD4 receptor bound. Furthermore, surface plasmon resonance (SPR) and enzyme-linked immunosorbance assays (ELISAs) showed that disorder was associated with poorer recognition by antibodies targeting conserved conformational epitopes. These data provide additional insight into the structural determinants of gp120 antigenicity and suggest that conformational dynamics should be considered in the selection and design of optimized Env immunogens.

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Section: Figsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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“…Other published experiments suggest a temperature-sensitive interaction with cell surface-expressed Env, but the structural heterogeneity of cleaved envelope protein on cell surfaces and the potential lipid-binding ability of 2F5 make those results difficult to interpret (25)(26)(27). The gp140 preparations reported to bind 2F5 all contain significant amounts of monomers, dimers, or aggregates (11,22).…”

Section: Stable Conformations Of Hiv Envelope Glycoprotein Gp140 Trimentioning

confidence: 98%

“…The stability of HIV-1 gp140 varies greatly from strain to strain; it can be enhanced by adding a C-terminal trimerization tag such as the T4-fibritin ''foldon'' or the coiled-coil trimer derived from GCN4 (11,12). Recombinant simian immunodeficiency virus (SIV) gp140 is a stable trimer even without such a tag (13).…”

Section: Stable Conformations Of Hiv Envelope Glycoprotein Gp140 Trimentioning

confidence: 99%

A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

Frey

Peng²,

Rits-Volloch³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

221

305

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Most antibodies induced by HIV-1 are ineffective at preventing initiation or spread of infection because they are either nonneutralizing or narrowly isolate-specific. Rare, ''broadly neutralizing'' antibodies have been detected that recognize relatively conserved regions on the envelope glycoprotein. Using stringently characterized, homogeneous preparations of trimeric HIV-1 envelope protein in relevant conformations, we have analyzed the molecular mechanism of neutralization by two of these antibodies, 2F5 and 4E10. We find that their epitopes, in the membrane-proximal segment of the envelope protein ectodomain, are exposed only on a form designed to mimic an intermediate state during viral entry. These results help explain the rarity of 2F5-and 4E10-like antibody responses and suggest a strategy for eliciting them. envelope glycoprotein ͉ membrane fusion H IV-1 infection generally induces a strong antibody response to the envelope glycoprotein [trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ], the sole antigen on the virion surface. Most induced antibodies are ineffective in preventing infection, however, because they are either nonneutralizing or narrowly isolate-specific, and the virus replicates so rapidly that ongoing selection of neutralization resistant mutants allows viral evolution to ''keep ahead'' of highaffinity antibody production (1). Moreover, much of the antibody response may be to rearranged or dissociated forms of gp120 and gp41, on which the dominant epitopes may be either in hypervariable loops or in positions occluded on virion-borne envelope trimer. Rare, ''broadly neutralizing'' antibodies have been detected that recognize one of three relatively conserved regions on the envelope protein: the CD4-binding site (mAb b12) (2); carbohydrates on the outer gp120 surface (mAb 2G12) (3); and a segment of the gp41 ectodomain adjacent to the viral membrane (mAbs 2F5 and 4E10) (4, 5), often called the ''membrane-proximal external region'' (MPER). We seek to understand the molecular mechanisms of neutralization by these and other antibodies.Fusion of viral and target-cell membranes initiates HIV-1 infection. Conformational changes in gp120 that accompany its binding to receptor (CD4) and coreceptor (e.g., CCR5 or CXCR4) lead to dissociation of gp120 from gp41 and a cascade of refolding events in the latter (6). In the course of these rearrangements, the N-terminal fusion peptide of gp41 translocates and inserts into the target-cell membrane. A proposed extended conformation of the gp41 ectodomain, with its fusion peptide thus inserted and the transmembrane anchor still in the viral membrane, has been called the ''prehairpin intermediate'' (7). It is the target of various fusion inhibitors, including T-20/enfuvirtide, the first approved fusioninhibiting antiviral drug (8), and the characteristics of the intermediate have been deduced from the properties of these inhibitors or mimicries by short gp41 fragments (9). Subsequent rearrangements from the intermediate to the postfusion state of gp41 involve ...

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“…Thus, like many other soluble Env preparations (12,29), it does not fully recapitulate the antigenic structure of virion-borne Env. Our recent observation that the TMD of HIV-1 Env is a structural component critical for the stability of the functional Env spike provides a plausible explanation for why the antigenic properties of most recombinant, soluble Env preparations with the TMD deleted deviate from those of the native protein (14,39). We therefore must take the structural constraints imposed by the TMD on the ectodomain into consideration when designing a soluble immunogen, e.g., by mutating surface-exposed, lipid-interacting hydrophobic residues on the TMD while maintaining the trimeric structure seen in the high-resolution structure (14,40).…”

Section: Discussionmentioning

confidence: 99%

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike

Cai

Karaca-Griffin

Chen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easyto-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160) 3 , retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.HIV-1 gp160 | neutralizing antibodies | vaccine design

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Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1

Cited by 48 publications

References 49 publications

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike

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