Conformational changes in env oligomer induced by an antibody dependent on the V3 loop base

Cavacini, Lisa A.; Duval, Mark; Song, Leslie; Sangster, Rebecca; Xiang, Shi Hua; Sodroski, Joseph; Posner, Marshall R.

doi:10.1097/00002030-200303280-00006

Cited by 48 publications

(47 citation statements)

References 30 publications

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“…2, the reactivity of recombinant Abs generated in CHO cells, including IgG1, IgG3, and IgG4, paralleled that observed for the parental F240 produced by hybridoma cells. F240 constructs were also tested for reactivity with primary isolate virions from clades B and C, some of which are considered either neutralization resistant or sensitive (34), and identical results were obtained (data not shown). Thus, CHO-derived F240 isotypes retained broad reactivity with gp41.…”

Section: Immunoreactivity Of Isotype Switched F240mentioning

confidence: 69%

“…Alternatively, for the isotype switch mutants the reporter readout was based on ␤-galactosidase production, which was measured by ELISA (Pierce). A PBMC-based assay (26,34) was also used to confirm HIV-1 neutralization in a more biologically relevant model. Briefly, serial 2-fold dilutions of a mAb were incubated with virus stock diluted to 200 50% tissue culture infective dose TCID 50 for 1 h at 37 o C before the addition of PHA-stimulated PBMC target cells (1 ϫ 10 5 cells/ well).…”

Section: Neutralization Assaysmentioning

confidence: 99%

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The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

Miranda

Duval

Doherty

et al. 2007

The Journal of Immunology

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Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.

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Section: Immunoreactivity Of Isotype Switched F240mentioning

confidence: 69%

Section: Neutralization Assaysmentioning

confidence: 99%

The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

Miranda

Duval

Doherty

et al. 2007

The Journal of Immunology

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“…S3). Enhancement of tyrosine sulfation by TPST2 overexpression dramatically reduced binding of monomeric sCD4 (receptor-binding site), mAb 412d (coreceptor-binding site) (37), and anti-V3 loop mAbs B4e8 (38) and D19 (39), all ligands with a restricted binding capacity to the native trimeric spike. In contrast, TPST2 overexpression markedly increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145, which are directed to quaternary, glycan-dependent V2 epitopes that are stabilized on the native trimer (22,23,40).…”

Section: Resultsmentioning

confidence: 99%

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Cimbro¹,

Gallant²,

Dolan

et al. 2014

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

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Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4 + T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.T he development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor-and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, l...

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“…The monoclonal antibodies used in this study were 17b, 48d, A32, C11, 19b, and 39F, kindly provided by J. Robinson (31,32,47,50,54); 447-52D and 697-D, kindly provided by M. Gorny and Susan Zolla-Pazner (12, 16-18); 8.22.2, kindly provided by A. Pinter (21); 2G12, kindly provided by H. Katinger (10,42,46,51); F425-B4e8, kindly provided by L. Cavacini (11); and HIV-IG, kindly provided by J. Mascola. MAbs b12, b6, b3, and 4KG5 were produced in-house (1, 2, 8, 9, 60); D7324 was purchased from Cliniqa (Fallbrook, CA), and sCD4 was purchased from Progenics (NY).…”

Section: Methodsmentioning

confidence: 99%

Comparing Antigenicity and Immunogenicity of Engineered gp120

Selvarajah

Puffer

Pantophlet

et al. 2005

J Virol

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We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein. Importantly, serum mapping showed that both mutants did not elicit antibodies against a number of epitopes that had been targeted for dampening. The sera from rabbits immunized with the GDMR gp120 mutant neutralized some primary viruses at levels somewhat better than the wild-type gp120 immune sera as a result of an increased elicitation of anti-V3 antibodies. Unlike wild-type gp120 immune sera, GDMR gp120 immune sera failed to neutralize HXBc2, a T-cell line adapted (TCLA) virus. This was associated with loss of CD4bs/CD4-induced antibodies that neutralize TCLA but not primary viruses. The mCHO gp120 immune sera did not neutralize primary viruses to any significant degree, reflecting the masking of epitopes of even weakly neutralizing antibodies without eliciting b12-like antibodies. These results show that antibody responses to multiple epitopes on gp120 can be dampened. More precise focusing to a neutralizing epitope will likely require several iterations comparing antigenicity and immunogenicity of engineered proteins.

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Conformational changes in env oligomer induced by an antibody dependent on the V3 loop base

Abstract: The conserved epitopes exposed by B4e8 are similar to those exposed by the movement of the variable loops following CD4 engagement. Further studies with select antibody combinations should provide important information for the design of effective immunotherapeutic agents.

Cited by 48 publications

References 30 publications

The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Comparing Antigenicity and Immunogenicity of Engineered gp120

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