Limits...
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.

Show MeSH

Related in: MedlinePlus

Native-PAGE band-shift and SEC-MALS analyses of coreS-antibody complexes.Interaction of (A) Fab8066, (B) Sc66 and (C) Sc62 with the six-helix bundle coreS antigen (Ag). Left panels: 10 µM coreS trimer mixed with Fab or ScFv (shown above the lanes) in molar ratios of 1∶1, 1∶2 and 1∶3 were subjected to 20% homogeneous native-PAGE. CoreS and antibody are color coded orange and blue, respectively. Calculated molecular weights of Fab8066, Sc66, Sc62, coreS and their complexes are indicated in kDa. Right panels: Protein mixtures (total of 200 µg) at a trimer (coreS) to antibody ratio of 1∶1 were subjected to SEC-MALS. Experimental average masses and compositions are indicated. Also shown in panel A (right) are the SEC-MALS traces for coreS alone (black), corresponding to a trimer (of calculated mass 3×8284 g/mol), and a 1∶1 mixture of coreS and Fab8062 (dashed gray) which shows no evidence of complex formation. The black arrows in panels B and C (right) indicate the retention volume of Sc66 (see Figure S2C in File S1). In panel C (right), the major peak corresponds to uncomplexed ScFv and coreS which co-elute, while the minor peak represents a barely detectable amount of 1∶1 Sc62:coreS complex. Observed masses (g/mol) for free Fab8066, Fab8062 and Sc66 are 47060±559, 48560±629 and 27630±774, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g003: Native-PAGE band-shift and SEC-MALS analyses of coreS-antibody complexes.Interaction of (A) Fab8066, (B) Sc66 and (C) Sc62 with the six-helix bundle coreS antigen (Ag). Left panels: 10 µM coreS trimer mixed with Fab or ScFv (shown above the lanes) in molar ratios of 1∶1, 1∶2 and 1∶3 were subjected to 20% homogeneous native-PAGE. CoreS and antibody are color coded orange and blue, respectively. Calculated molecular weights of Fab8066, Sc66, Sc62, coreS and their complexes are indicated in kDa. Right panels: Protein mixtures (total of 200 µg) at a trimer (coreS) to antibody ratio of 1∶1 were subjected to SEC-MALS. Experimental average masses and compositions are indicated. Also shown in panel A (right) are the SEC-MALS traces for coreS alone (black), corresponding to a trimer (of calculated mass 3×8284 g/mol), and a 1∶1 mixture of coreS and Fab8062 (dashed gray) which shows no evidence of complex formation. The black arrows in panels B and C (right) indicate the retention volume of Sc66 (see Figure S2C in File S1). In panel C (right), the major peak corresponds to uncomplexed ScFv and coreS which co-elute, while the minor peak represents a barely detectable amount of 1∶1 Sc62:coreS complex. Observed masses (g/mol) for free Fab8066, Fab8062 and Sc66 are 47060±559, 48560±629 and 27630±774, respectively.

Mentions: ITC measurements on the interactions of Fabs and ScFvs with the three six-helix bundle mimetics displayed negligible thermal responses. We therefore resorted to optimize a method to monitor antigen-antibody binding for these various constructs by native polyacrylamide gel electrophoresis (native-PAGE) and analytical size-exclusion chromatography with in-line multiangle light scattering, refractive index and UV detectors (SEC-MALS) (Fig. 3).


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)

Native-PAGE band-shift and SEC-MALS analyses of coreS-antibody complexes.Interaction of (A) Fab8066, (B) Sc66 and (C) Sc62 with the six-helix bundle coreS antigen (Ag). Left panels: 10 µM coreS trimer mixed with Fab or ScFv (shown above the lanes) in molar ratios of 1∶1, 1∶2 and 1∶3 were subjected to 20% homogeneous native-PAGE. CoreS and antibody are color coded orange and blue, respectively. Calculated molecular weights of Fab8066, Sc66, Sc62, coreS and their complexes are indicated in kDa. Right panels: Protein mixtures (total of 200 µg) at a trimer (coreS) to antibody ratio of 1∶1 were subjected to SEC-MALS. Experimental average masses and compositions are indicated. Also shown in panel A (right) are the SEC-MALS traces for coreS alone (black), corresponding to a trimer (of calculated mass 3×8284 g/mol), and a 1∶1 mixture of coreS and Fab8062 (dashed gray) which shows no evidence of complex formation. The black arrows in panels B and C (right) indicate the retention volume of Sc66 (see Figure S2C in File S1). In panel C (right), the major peak corresponds to uncomplexed ScFv and coreS which co-elute, while the minor peak represents a barely detectable amount of 1∶1 Sc62:coreS complex. Observed masses (g/mol) for free Fab8066, Fab8062 and Sc66 are 47060±559, 48560±629 and 27630±774, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4126735&req=5

pone-0104683-g003: Native-PAGE band-shift and SEC-MALS analyses of coreS-antibody complexes.Interaction of (A) Fab8066, (B) Sc66 and (C) Sc62 with the six-helix bundle coreS antigen (Ag). Left panels: 10 µM coreS trimer mixed with Fab or ScFv (shown above the lanes) in molar ratios of 1∶1, 1∶2 and 1∶3 were subjected to 20% homogeneous native-PAGE. CoreS and antibody are color coded orange and blue, respectively. Calculated molecular weights of Fab8066, Sc66, Sc62, coreS and their complexes are indicated in kDa. Right panels: Protein mixtures (total of 200 µg) at a trimer (coreS) to antibody ratio of 1∶1 were subjected to SEC-MALS. Experimental average masses and compositions are indicated. Also shown in panel A (right) are the SEC-MALS traces for coreS alone (black), corresponding to a trimer (of calculated mass 3×8284 g/mol), and a 1∶1 mixture of coreS and Fab8062 (dashed gray) which shows no evidence of complex formation. The black arrows in panels B and C (right) indicate the retention volume of Sc66 (see Figure S2C in File S1). In panel C (right), the major peak corresponds to uncomplexed ScFv and coreS which co-elute, while the minor peak represents a barely detectable amount of 1∶1 Sc62:coreS complex. Observed masses (g/mol) for free Fab8066, Fab8062 and Sc66 are 47060±559, 48560±629 and 27630±774, respectively.
Mentions: ITC measurements on the interactions of Fabs and ScFvs with the three six-helix bundle mimetics displayed negligible thermal responses. We therefore resorted to optimize a method to monitor antigen-antibody binding for these various constructs by native polyacrylamide gel electrophoresis (native-PAGE) and analytical size-exclusion chromatography with in-line multiangle light scattering, refractive index and UV detectors (SEC-MALS) (Fig. 3).

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
Related in: MedlinePlus