Structure-Function Analysis of the Epitope for 4E10, a Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody

epartments of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., CVN-6, La Jolla, CA 92037, USA.
Journal of Virology (Impact Factor: 4.44). 02/2006; 80(4):1680-7. DOI: 10.1128/JVI.80.4.1680-1687.2006
Source: PubMed


The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the K(d) values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH(2) could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope.

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    • "The 2F5 and 4E10 epitopes are naturally adjacent in the context of HIV-1, offering the hope that their joint inclusion might enhance our ability to present them in native-like conformations and to maximize the opportunities for eliciting an effective immune response. The 4E10 epitope consists largely of the tryptophan-rich, contiguous sequence N671WF(D/N) ITNWLW680 [33,62,63,64,65] and is recognized by the 4E10 mAb, one of the most broadly neutralizing anti-HIV antibodies, capable of neutralizing virus isolates from all group M HIV-1 subtypes [21,63,66,67]. Crystallographic and NMR studies have demonstrated that the 4E10 epitope adopts an amphipathic helical conformation, significantly embedded in the viral membrane and varying in the extent to which it is slightly bent or straight in the middle of the epitope, depending on the constructs and conditions used [29,33,57,65,68]. "
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    ABSTRACT: The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV. Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested. Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.
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    • "A number of groups have isolated several monoclonal antibodies (mAbs) that can neutralize a variety of HIV-1 isolates [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]. Among those mAbs, 2F5, 4E10, Z13 and the recently reported 10E8 are shown to target conserved epitopes within the membraneproximal external region (MPER) of gp41 [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]; The isolation of these and other broadly neutralizing antibodies (b12, 2G12, VRC01, CH01 to CH04, PG9, PG16) is a milestone in the field. The epitopes located at the MPER of gp41 recognized by broadly neutralizing mAbs 2F5 and 4E10 are promising targets for vaccine design. "
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    • "TZM-bl cells [79] were obtained from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. via the NIH ARRRP. Production of HIV-1 PSVs and TZM-bl neutralization assays were carried out as described previously [80]. To produce virus in the presence of kifunensine (kif), 25 µM kif (Sigma-Aldrich) was added to the 293T cell culture media immediately prior to transfection. "
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