Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 24;10(7):e1004271.
doi: 10.1371/journal.ppat.1004271. eCollection 2014 Jul.

Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization

Arthur S Kim  1 , Daniel P Leaman  1 , Michael B Zwick  1
Affiliations

Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization

Arthur S Kim et al. PLoS Pathog. .

Abstract

Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Partial neutralization of HIV-1 JR2, SF162 and corresponding MPER mutants by 10E8.
(A) Diagram of the MPER and surrounding domains of HIV-1 gp41 with residues found to be important for neutralization by 10E8 highlighted in red. (B) Neutralization of JR2 and cognate MPER mutants against broadly neutralizing antibody 10E8 (left) performed side-by-side with 4E10 (middle) and 2F5 (right). (C) Neutralization of SF162 wild type and F673 mutants by 10E8. (D) Maximum neutralization (plateau) percentages and (E) dose response neutralization curves of 10E8 (IgG and Fab) against JR2 and SF162 wild type and F673 Ala mutants. Error bars are from two independent experiments [n = 2] performed in duplicate using TZM-bl target cells.
Figure 2
Figure 2. Presence of 10E8 diminishes neutralization sensitivity of HIV-1 to 2F5 and Z13e1.
The maximum infectivity of virus in presence or absence of 10E8 was normalized to 100% for mutant F673L (left panels) and wild type HIV-1 (right panels); 10E8 was held constant at 10 µg/ml and the IC50 for F673L mutants and wild type viruses, respectively. The 10E8 resistant fraction of viral infectivity was assayed against (A and C) 2F5 and (B and D) Z13e1. Neutralization curves in black and red indicate absence and presence of 10E8, respectively, for (A and B) SF162 and (C and D) JR-FL. Experiments were performed in duplicate in at least two independent experiments, except for wild type SF162 that was tested in four independent experiments (n = 4). Statistical significance was determined for each pair of points along the dose response curves (in presence or absence of 10E8) using an unpaired t test and p-values were corrected using the Sidak-Bonferroni method for multiple comparisons. Symbols above each pair of data points represent the p-values (*, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant). (E) Statistical significance of the change in IC50 of 2F5 against JR2 F673L due to the presence of 10E8 was determined using linear regression analysis.
Figure 3
Figure 3. Glycosylation state of MPER mutant HIV-1 Env influences extent of maximum neutralization by 10E8.
The JR2 F673L mutant was subjected to various glycosylation conditions and the corresponding viruses were tested in a neutralization assay against 10E8 using TZM-bl target cells to determine the maximum neutralization percentage.
Figure 4
Figure 4. Effect of presence of 10E8 on the sensitivity of HIV-1 and corresponding F673L mutant to various ligands (IC50).
Asterisk (*) indicates fold change in IC50 was calculated using the equation: (IC50 without 10E8/IC50 with 10E8). Purple highlight shows when neutralization potency is decreased by greater than three fold and gold highlight shows when neutralization potency is increased greater than three fold. Hashtag (#) indicates that the data is not applicable (na) as an IC50 was not reached at concentrations tested.
Figure 5
Figure 5. Presence of 10E8 alters sensitivity of HIV-1 to soluble CD4 as well as to antibodies against CD4 and coreceptor binding sites.
The presence (red) or absence (black) of 10E8 at 10 µg/ml or the IC50 for F673L mutants and wild type viruses, respectively, affects the neutralization sensitivity of cognate virus to (A) soluble CD4, (B) b6 IgG and (C) 17b IgG.
Figure 6
Figure 6. Presence of 10E8 alters sensitivity of HIV-1 to post-attachment fusion inhibitors.
The presence (red) or absence (black) of 10E8 at 10 µg/ml or the IC50 for F673L mutants and wild type viruses, respectively, affects the neutralization sensitivity of cognate virus to (A) DN9 IgG and (B) C34 peptide.
Figure 7
Figure 7. 10E8 binds less efficiently to Env spikes of MPER mutant F673A compared to wild type.
Fab 10E8 was incubated with JR-FL (A) wild type or (B) F673A virions. In washout experiments, virion-Fab mixtures were pelleted by centrifugation and supernatants replaced with buffer devoid of Fab. Gel mobility of Env trimers was determined using BN-PAGE and Western blot probed with a cocktail of gp120 and gp41 antibodies. (C) BN-PAGE gel mobility shift data were used to generate binding curves for 10E8 against detergent-solubilized Env spikes. The intensity of Env trimer band that remained unshifted at each concentration of 10E8 relative to that of an antibody-free control was determined using densitometry for eight independent blots and averaged results were plotted. (D) Occupancy of 10E8 binding to Env trimers at different concentrations was quantified by measuring the distance between midpoints of Fab-shifted vs untreated bands, and plotting the result as a function of the distance shifted by Fabs b12 and PG9 that are assumed to bind three and one Fab(s) per trimer, respectively. Eight independent blots were analyzed for the “no washout” condition and three independent blots for the “washout” treatment. The averaged data points do not fall on whole numbers so a line is shown at the nearest round number of Fab occupancy to facilitate interpretation. (E) 10E8, 4E10 or Z13e1 Fabs were incubated with JR-FL wild type or F673A virions and resulting changes to gel mobility of Env trimers was determined using BN-PAGE and Western blot. A cocktail of gp120 and gp41 antibodies were used to probe the blot. Electrophoresis was performed using 3–8% Tris-Acetate NuPAGE gels (Invitrogen) under native conditions. Stoichiometry of binding was determined by measuring the gel mobility shift compared to Fab controls b12 and PG9.
Figure 8
Figure 8. Enhancement by 10E8 of the functional stability of MPER mutant F673L and corresponding diminution of functional stability of wild type HIV-1 Env.
(A) Half-life (t1/2) of infectivity decay at 37°C and (B) thermostability of JR2 wild type and (C and D, respectively) F673L mutant in the presence of incremental concentrations of 10E8. Thermostability was determined using a heat gradient to obtain the temperature at which viral infectivity drops by 90% (T90). (E) Relationship of 10E8-induced changes in infectivity decay (Δt1/2) of JR2 F673L mutant is plotted against that of wild type JR2. (F) The 10E8-induced change in thermostability (ΔT90) of wild type JR2 is inversely correlated with that of the F673L mutant.
Figure 9
Figure 9. Presence of 10E8 enhances functional thermostability of multiple MPER mutants of JR2 and SF162 within the 10E8 linear epitope.
(A) The presence of 10E8 at a concentration in the maximum neutralization plateau (10 µg/ml) enhances functional thermostability (T90) of multiple MPER mutants of JR2. Hash tag (#) indicates insufficient infectivity. (B) The effect on the functional thermostability of JR2 F673L by the presence of control antibodies 4E10, Z13e1 and DEN3, which are non-neutralizing, neutralizing and irrelevant antibodies against this mutant, respectively. 4E10 and Z13e1 were used at 50 µg/ml and DEN3 was used at 100 µg/ml. (C) Functional thermostability of SF162 in the presence of 10E8 at a concentration in the maximum neutralization plateau (10 µg/ml).
Figure 10
Figure 10. 10E8 enhances physical stability of Env trimers against heat-induced denaturation.
JR-FL (A) wild type and (B) F673A mutant virions were exposed to increasing temperatures in the absence (leftmost lanes) or presence of 100 µg/ml 10E8 Fab (rightmost lanes). Following detergent solubilization, the oligomeric state of Env was determined using BN-PAGE and Western blot probed with anti-gp41 antibodies. The intensity of the Env trimer bands from each lane were quantified and plotted as the percentage of trimer remaining relative to that incubated at 37°C (far right). (C) JR-FL wild type or F673A virions or (D) detergent-solubilized Env was incubated in the presence or absence of 10E8 Fab over a time course at 37°C, and the oligomeric state of Env was analyzed using BN-PAGE Western blot.
Figure 11
Figure 11. Kinetics of neutralization of JR2 F673L mutant by 10E8 and C34.
(A) Wild type JR2 and (B) mutant F673L were incubated with MPER antibodies or fusion inhibitor C34 to the NHR of gp41 over a time course at 37°C before adding onto target (TZM-bl) cells.
Figure 12
Figure 12. Functional effects of 10E8 on HIV-1 clade C variants including those with a naturally occurring L673 residue.
(A) Partial neutralization by 10E8 of clade C isolates TM20.13 and M20490 BMR 211 using the TZM-bl assay. (B) Effect of the presence of 10E8 at a saturating concentration (10 µg/ml) on thermostability (T90) of clade C isolates or cognate F673 mutants. Hash tag (#) indicates insufficient infectivity due to lability or neutralization. (C) Percentage of maximum neutralization by 10E8 of various clade C isolates or cognate F673 mutants. (D) Effect of the presence of 10E8 on neutralization sensitivity of clade C isolates or cognate F673 mutants to various ligands. Fold change in IC50 was calculated using the equation: (IC50 without 10E8/IC50 with 10E8). Purple highlight shows when neutralization potency is decreased by greater than three-fold and gold highlight shows when neutralization potency is increased greater than three-fold. nd, not determined.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Burton DR, Ahmed R, Barouch DH, Butera ST, Crotty S, et al. (2012) A Blueprint for HIV Vaccine Discovery. Cell Host Microbe 12: 396–407. - PMC - PubMed
    1. Mascola JR, Haynes BF (2013) HIV-1 neutralizing antibodies: understanding nature's pathways. Immunol Rev 254: 225–244. - PMC - PubMed
    1. Klein F, Mouquet H, Dosenovic P, Scheid JF, Scharf L, et al. (2013) Antibodies in HIV-1 vaccine development and therapy. Science 341: 1199–1204. - PMC - PubMed
    1. Agrawal N, Leaman DP, Rowcliffe E, Kinkead H, Nohria R, et al. (2011) Functional stability of unliganded envelope glycoprotein spikes among isolates of human immunodeficiency virus type 1 (HIV-1). PLoS One 6: e21339. - PMC - PubMed
    1. Layne SP, Merges MJ, Dembo M, Spouge JL, Conley SR, et al. (1992) Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus. Virology 189: 695–714. - PubMed

Publication types

MeSH terms

Substances