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Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

Louis JM, Aniana A, Lohith K, Sayer JM, Roche J, Bewley CA, Clore GM - PLoS ONE (2014)

Bottom Line: Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity.The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs.We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

View Article: PubMed Central - PubMed

Affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

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Determining the upper limit of affinity for the binding of Fab8066 to coreS by native-PAGE and an approximate KD by SEC-MALS.(A) Decreasing concentrations (10 to 1.25 µM) of coreS trimer mixed with a 2-fold molar excess of Fab8066 and subjected to native-PAGE with Coomassie staining (upper panel), and decreasing concentrations (1 to 0.25 µM) of coreS trimer mixed with a constant 1 µM concentration of Fab8066, visualized by silver staining. CoreS and Fab8066 are color coded orange and blue, respectively. (B) Injection of 3 µg coreS mixed with 6 µg Fab8066 on a BioSep-SEC-S 2000 column (0.46×30 cm) at a flow-rate of 0.35 ml/min equilibrated in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl (buffer A). The elution profile (black) is shown superimposed on deconvoluted peaks for the major (∼85%) coreS-Fab8066 complex (red) and free Fab8066 (blue). The measured mass of the complex is shown beside the peak. A KD of ∼70 nM was estimated on the basis of the calculated concentration of the complex and free Fab. Deconvolution of the SEC-MALS profile was carried out using the program PeakFit (Seasolve Software, Inc. Framingham, MA).
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pone-0104683-g006: Determining the upper limit of affinity for the binding of Fab8066 to coreS by native-PAGE and an approximate KD by SEC-MALS.(A) Decreasing concentrations (10 to 1.25 µM) of coreS trimer mixed with a 2-fold molar excess of Fab8066 and subjected to native-PAGE with Coomassie staining (upper panel), and decreasing concentrations (1 to 0.25 µM) of coreS trimer mixed with a constant 1 µM concentration of Fab8066, visualized by silver staining. CoreS and Fab8066 are color coded orange and blue, respectively. (B) Injection of 3 µg coreS mixed with 6 µg Fab8066 on a BioSep-SEC-S 2000 column (0.46×30 cm) at a flow-rate of 0.35 ml/min equilibrated in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl (buffer A). The elution profile (black) is shown superimposed on deconvoluted peaks for the major (∼85%) coreS-Fab8066 complex (red) and free Fab8066 (blue). The measured mass of the complex is shown beside the peak. A KD of ∼70 nM was estimated on the basis of the calculated concentration of the complex and free Fab. Deconvolution of the SEC-MALS profile was carried out using the program PeakFit (Seasolve Software, Inc. Framingham, MA).

Mentions: Since we were unable to successfully use ITC to quantitatively determine the binding affinities of our neutralizing antibodies in either Fab or ScFv formats to the six-helix bundle mimetics, we used native-PAGE band-shift analysis to obtain a semi-quantitative upper limit for the strength of the interaction of Fab8066 with coreS. Fab8066 rather than Sc66 was used in this assay since the larger size of Fab8066 improves detectability on gel staining and Fab8066 exclusively forms a 1∶1 complex with coreS rather than the mixture of 1∶1 and 1∶3 antigen:Fab complexes observed with Sc66. In the first set of native-PAGE band-shift experiments we prepared 1∶2 mixtures of coreS trimer with Fab8066 in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl at Fab concentrations ranging from 10 to 1.25 µM. As the limit of detectability with Coomassie staining was reached at 1.25 µM of the coreS-Fab8066 1∶1 complex (Fig. 6A, top), silver staining was used to provide improved detection at lower concentrations. A set of dilutions (Fig. 6A, bottom) was made in which Fab8066 at a constant concentration of 1 µM was titrated by addition of coreS to give final concentrations ranging from 1 µM to 0.25 µM coreS. The increase in band intensity of the 1∶1 complex, accompanied by a gradual decrease in intensity of free Fab8066, in going from 0.25 to 1 µM coreS shows that complex formation is accompanied by depletion of Fab8066. At the lowest concentration of coreS only a small amount of the 1∶1 complex can be detected. This serves to set an upper limit of KD≤250 nM for the binding of Fab8066 to coreS. By way of contrast, a lower limit of KD>>10 µM for the binding of Fab8062 and Sc62 to coreS can be estimated from the SEC-MALS and native-PAGE data shown in Figs. 3 A and C, respectively.


Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

Louis JM, Aniana A, Lohith K, Sayer JM, Roche J, Bewley CA, Clore GM - PLoS ONE (2014)

Determining the upper limit of affinity for the binding of Fab8066 to coreS by native-PAGE and an approximate KD by SEC-MALS.(A) Decreasing concentrations (10 to 1.25 µM) of coreS trimer mixed with a 2-fold molar excess of Fab8066 and subjected to native-PAGE with Coomassie staining (upper panel), and decreasing concentrations (1 to 0.25 µM) of coreS trimer mixed with a constant 1 µM concentration of Fab8066, visualized by silver staining. CoreS and Fab8066 are color coded orange and blue, respectively. (B) Injection of 3 µg coreS mixed with 6 µg Fab8066 on a BioSep-SEC-S 2000 column (0.46×30 cm) at a flow-rate of 0.35 ml/min equilibrated in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl (buffer A). The elution profile (black) is shown superimposed on deconvoluted peaks for the major (∼85%) coreS-Fab8066 complex (red) and free Fab8066 (blue). The measured mass of the complex is shown beside the peak. A KD of ∼70 nM was estimated on the basis of the calculated concentration of the complex and free Fab. Deconvolution of the SEC-MALS profile was carried out using the program PeakFit (Seasolve Software, Inc. Framingham, MA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g006: Determining the upper limit of affinity for the binding of Fab8066 to coreS by native-PAGE and an approximate KD by SEC-MALS.(A) Decreasing concentrations (10 to 1.25 µM) of coreS trimer mixed with a 2-fold molar excess of Fab8066 and subjected to native-PAGE with Coomassie staining (upper panel), and decreasing concentrations (1 to 0.25 µM) of coreS trimer mixed with a constant 1 µM concentration of Fab8066, visualized by silver staining. CoreS and Fab8066 are color coded orange and blue, respectively. (B) Injection of 3 µg coreS mixed with 6 µg Fab8066 on a BioSep-SEC-S 2000 column (0.46×30 cm) at a flow-rate of 0.35 ml/min equilibrated in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl (buffer A). The elution profile (black) is shown superimposed on deconvoluted peaks for the major (∼85%) coreS-Fab8066 complex (red) and free Fab8066 (blue). The measured mass of the complex is shown beside the peak. A KD of ∼70 nM was estimated on the basis of the calculated concentration of the complex and free Fab. Deconvolution of the SEC-MALS profile was carried out using the program PeakFit (Seasolve Software, Inc. Framingham, MA).
Mentions: Since we were unable to successfully use ITC to quantitatively determine the binding affinities of our neutralizing antibodies in either Fab or ScFv formats to the six-helix bundle mimetics, we used native-PAGE band-shift analysis to obtain a semi-quantitative upper limit for the strength of the interaction of Fab8066 with coreS. Fab8066 rather than Sc66 was used in this assay since the larger size of Fab8066 improves detectability on gel staining and Fab8066 exclusively forms a 1∶1 complex with coreS rather than the mixture of 1∶1 and 1∶3 antigen:Fab complexes observed with Sc66. In the first set of native-PAGE band-shift experiments we prepared 1∶2 mixtures of coreS trimer with Fab8066 in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl at Fab concentrations ranging from 10 to 1.25 µM. As the limit of detectability with Coomassie staining was reached at 1.25 µM of the coreS-Fab8066 1∶1 complex (Fig. 6A, top), silver staining was used to provide improved detection at lower concentrations. A set of dilutions (Fig. 6A, bottom) was made in which Fab8066 at a constant concentration of 1 µM was titrated by addition of coreS to give final concentrations ranging from 1 µM to 0.25 µM coreS. The increase in band intensity of the 1∶1 complex, accompanied by a gradual decrease in intensity of free Fab8066, in going from 0.25 to 1 µM coreS shows that complex formation is accompanied by depletion of Fab8066. At the lowest concentration of coreS only a small amount of the 1∶1 complex can be detected. This serves to set an upper limit of KD≤250 nM for the binding of Fab8066 to coreS. By way of contrast, a lower limit of KD>>10 µM for the binding of Fab8062 and Sc62 to coreS can be estimated from the SEC-MALS and native-PAGE data shown in Figs. 3 A and C, respectively.

Bottom Line: Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity.The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs.We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

View Article: PubMed Central - PubMed

Affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Show MeSH