<i>Escherichia coli</i>Expression, Purification, and Biological Activity of a Truncated Soluble CD4

Garlick, Robert; Kirschner, Richard J.; Eckenrode, Frances M.; Tarpley, W. G.; Tomich, C.‐S. C.

doi:10.1089/aid.1990.6.465

Cited by 56 publications

(45 citation statements)

References 30 publications

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“…Antibodies IgG1-b12 (51-54), 17b (18,35,(55)(56)(57), VRC01 (58), A32 (35,59), and soluble two-domain CD4 (60) and CD4-IgG2 were obtained from the NIH AIDS Reagents Program. The antibodies N5i5, B18, and C4 were generously provided by Yongjun Guan and George Lewis (University of Maryland) (61,62).…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…HEK293 cells were obtained from the American Type Culture Collection. Soluble human D1D2 CD4 was obtained through the NIH AIDS Reference and Reagent Program, Division of AIDS, NIAID, NIH (18). The monoclonal antibodies b12 and b6 used in this study were engineered as immunoglobulin G1 constructs as previously described (6) and were generous gifts from Dennis Burton, and F105 (34,35) was kindly provided by Marshal Posner.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Human Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-Bound State: Antigenicity, Biophysics, and Immunogenicity

Dey

Pancera

Svehla

et al. 2007

J Virol

100

150

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The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S؉S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S؉S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

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“…When infection was performed in the presence of lactose, the sodium chloride concentration in the medium was adjusted to maintain appropriate osmolarity (i.e., 317 mosmol/L) (13). Inhibition studies were performed with the anti-CD4 mAb SIM.2 and soluble CD4 (sCD4-183) (35) and Pharmacia, respectively. Anti-HIV-1 IgG Abs consist of a pool of affinity column-purified serum from three HIV-1-infected patients, which has been observed to bind to many viral components, including gp120 (our unpublished data).…”

Section: Infection Studiesmentioning

confidence: 99%

Galectin-1 Acts as a Soluble Host Factor That Promotes HIV-1 Infectivity through Stabilization of Virus Attachment to Host Cells

Ouellet¹,

Mercier²,

Pelletier

et al. 2005

The Journal of Immunology

155

161

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The establishment of HIV type 1 (HIV-1) infection is initiated by the stable attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between virus-encoded gp120 and cell surface CD4, a number of distinct interactions influence binding of HIV-1 to host cells. In this study, we report that galectin-1, a dimeric β-galactoside-binding protein, promotes infection with R5, X4, and R5X4 variants. Galectin-1 acts as a soluble adhesion molecule by facilitating attachment of HIV-1 to the cell surface. This postulate is based on experiments where galectin-1 rendered HIV-1 particles more refractory to various agents that block HIV-1 adsorption and coreceptor binding (i.e., a blocking anti-CD4, soluble CD4, human anti-HIV-1 polyclonal Abs; stromal cell-derived factor-1α; RANTES). Experiments performed with the fusion inhibitor T-20 confirmed that galectin-1 is primarily affecting HIV-1 attachment. The relevance of the present findings for the pathogenesis of HIV-1 infection is provided by the fact that galectin-1 is abundantly expressed in the thymus and lymph nodes, organs that represent major reservoirs for HIV-1. Moreover, galectin-1 is secreted by activated CD8+ T lymphocytes, which are found in high numbers in HIV-1-positive patients. Therefore, it is proposed that galectin-1, which is released in an exocrine fashion at HIV-1 replication sites, can cross-link HIV-1 and target cells and promote a firmer adhesion of the virus to the cell surface, thereby augmenting the efficiency of the infection process. Overall, our findings suggest that galectin-1 might affect the pathogenesis of HIV-1 infection.

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Escherichia coliExpression, Purification, and Biological Activity of a Truncated Soluble CD4

Cited by 56 publications

References 30 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

Characterization of Human Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-Bound State: Antigenicity, Biophysics, and Immunogenicity

Galectin-1 Acts as a Soluble Host Factor That Promotes HIV-1 Infectivity through Stabilization of Virus Attachment to Host Cells

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