4E10 mAbs Research Articles

Anti-Idiotypic Monobodies Derived from a Fibronectin Scaffold

Mimetics of conformational protein epitopes have broad applications but have been difficult to identify using conventional peptide phage display. The 10th type III domain of human fibronectin (FNfn10) has two extended, randomizable surface-exposed loops and might be more amenable to the identification of such mimetics. We therefore selected a library of FNfn10 clones, randomized in both loops (15 residues in all), for binding to monoclonal antibodies (mAbs) that recognize the HIV-1 envelope glycoprotein. Anti-idiotypic monobodies (αIMs) mimicking both "linear" epitopes (2F5 and 4E10 mAbs) and conformational epitopes (b12 and VRC01 mAbs) were generated. αIMs selected against 2F5 and 4E10 frequently displayed sequence homology to the corresponding linear native epitopes. In the case of b12 and VRC01, we expected that the two constrained loop domains of FNfn10 would both contribute to complex conformational interactions with target antibodies. However, mutagenesis studies revealed differences from this simple model. An αIM selected against b12 was found to bind its cognate antibody via only a few residues within the BC loop of FNfn10, with minimal contribution from the FG loop. Unexpectedly, this was sufficient to generate a protein that engaged its cognate antibody in a manner very similar to that of HIV-1 Env, and with a strong KD (43 nM). In contrast, an αIM selected against VRC01 engaged its cognate antibody in a manner that was dependent on both BC and FG loop sequences. Overall, these data suggest that the FNfn10 scaffold can be used to identify complex structures that mimic conformational protein epitopes.

Neutralization and enhancement of trans infection by erythrocyte-bound HIV with antibodies and complement

Background Antibodies to HIV envelope glycoproteins, with or without neutralization when assayed in standard neutralization assays, are reported to have potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Although most of the standard neutralization assays use free virus rather than carrier-bound HIV as the infectious agent; a cell line as the target for infection; and no complement (C), these conditions may not reflect in vivo events that would include antibody-dependent innate effector mechanisms. Methods We investigated the Fc-receptor-mediated and the complement-mediated antibody-dependent enhancement in trans infection neutralization assays using two broadly neutralizing monoclonal antibodies, 4E10 and b12; primary HIV; PBMC target cells naturally expressing Fc receptors and complement receptors; and with or without complement. Results In the absence of complement, the 4E10 mAb did not neutralize erythrocyte-bound HIV, and the b12 mAb neutralized erythrocyte-bound HIV less effective than the free virus. At low concentrations, 4E10 caused enhancement of infection. In contrast, in the presence of complement, 4E10 neutralized erythrocyte-bound HIV, or caused enhancement of trans infection of erythrocytebound HIV, depending on the mechanism of binding of HIV to erythrocytes. Conclusion Our results are consistent with the concept that the binding of HIV to erythrocytes represents a mechanism of establishing a “safe harbor” for HIV from the adverse effects of antibodies and complement that might otherwise be detrimental to free circulating HIV. Our data shows that broadly neutralizing antibody 4E10 can cause both C-mediated neutralization and enhancement of trans infection. This suggests that erythrocyte-bound HIV-1 serves as a sort of battleground between neutralization and enhancement by antibodies in the presence of C. Because of this, we propose that induction of neutralizing vs. enhancing antibodies can only be differentiated by utilization of a trans infection neutralization assay that might yield more relevant results for in vivo conditions.

A new paradigm in molecular recognition? specific antibody binding to membrane-inserted HIV-1 epitopes

The conserved membrane proximal external region (MPER), adjacent to the transmembrane domain (TMD) of human immunodeficiency virus type-1 (HIV-1) gp41 glycoprotein subunit, is accessible to the broadly neutralizing 4E10 and 2F5 monoclonal antibodies (mAbs) and, therefore, constitutes a potential target for vaccine design. This gp41 domain is postulated to be functional during the Env glycoprotein-mediated fusion reaction by destabilizing the highly rigid viral envelope. To perform this task, the aromatic-rich MPER is believed to insert into the interfacial region of the viral membrane external monolayer, thereby inducing the restructuring of the lipid bilayer required for fusion-pore opening. This model predicts that: (i) 2F5 and 4E10 mAbs are capable of binding epitopes inserted into the membrane interface; (ii) in-membrane binding will result in effective blocking of MPER membrane activity; and (iii) both processes, in-membrane recognition and blocking of membrane activity, can be modulated by altering both the lipid composition and the MPER amino acid sequence. We review here recently reported experimental data consistent with those predictions, and further speculate on their relevance for prospective anti-HIV vaccine development.

The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies

Two human monoclonal anti-HIV-1 antibodies, 2F5 and 4E10, were utilized to investigate the accessibility and conservation of gp41 MPER epitopes on five different clades of HIV-1 in the absence and presence of sCD4. The binding of human monoclonal antibodies (mAbs) to HIV-1 was dependent upon the virus clade. Soluble CD4 significantly increased the accessibility of gp41 MPER-binding epitopes on several isolates that previously showed little or no binding with 2F5 and 4E10 mAbs as determined by a modified ELISA-based virus capture assay and surface plasmon resonance. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus. However, neither binding per se nor the total envelope content per virion was a predictor of neutralization. The hidden or conformational gp41 MPER epitopes unmasked by sCD4 may provide additional targets for vaccine design.

Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution

The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13, and 4E10 monoclonal antibodies (mAbs). One strategy to elicit such antibodies is to design an immunogen with increased exposure of the 2F5 and 4E10 mAb epitopes. In this study we characterize a single leucine to serine substitution at position 669 (L669S) in the gp41 Env MPER that confers >250-fold more neutralization sensitivity to 2F5 and 4E10 mAbs than does the wild-type gp41 sequence. On synthetic liposomes, increased solvent exposure of MPER tryptophan residues and stable docking of 2F5 and 4E10 mAbs to mutant MPER peptide liposomes indicate more favorable membrane orientation of MPER neutralizing epitopes with L669S substitution. The time during which virus is sensitive to 2F5 mAb-mediated neutralization is approximately 3-fold longer when the mutation is present. These data suggest that a major contribution to the L669S mutant virus phenotype of enhanced susceptibility to MPER mAbs is prolonged exposure of the MPER neutralizing epitope during viral entry.

OA021-04. HIV-1 gp41 envelope MPER mutation altered epitope conformation in lipid and increased sensitivity to 2F5 and 4E10 neutralizing antibodies

Background The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13 and 4E10 monoclonal antibodies (mAbs). However, MPER antibodies are rarely found in HIV-1 infected subjects nor arise following envelope immunization. A potential strategy to elicit such antibodies more frequently is to design an envelope protein with increased exposure of the 2F5 and 4E10 mAb epitopes.

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 4E10 Monoclonal Antibody Is Better Adapted to Membrane-Bound Epitope Recognition and Blocking than 2F5

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.

The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.

Simian Immunodeficiency Virus Engrafted with Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Epitopes: Replication, Neutralization, and Survey of HIV-1-Positive Plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.