4E10-resistant HIV-1 isolated from four subjects with rare membrane-proximal external region polymorphisms

Abstract

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope.

Figure 1. Maximum-Likelihood phylogenetic tree of all env sequences used in this study.

HXB2 is used as an outgroup to root the tree.

Figure 2. Alignment of MPER sequence variants from all subjects described in this study.

Sequences are aligned against HXB2 and a subtype C consensus sequence derived from the 2007 LANL database. Numbering is based on HXB2. MSD denotes start of the Membrane Spanning Domain. Consensus residues are color-coded by degree of conservation (red >98%, orange 90–97.9%, yellow 75–89.9%, green <75%). In panel A, IC₅₀ for mAb 4E10 is expressed as a range for all functional clones tested for each MPER variant. Positions 677, 680, 683, and 686 are color-coded for emphasis. In Panel B, Env protein sequences have been color-coded according to charge at positions 677, 680, and 683 (blue = basically charged residue, yellow = uncharged residue). All other residues (in white) are uncharged.

Figure 3. Binding ELISA results for subject 16Ms plasma.

Binding ELISA testing subject 16Ms plasma against normal human plasma and four monoclonal antibodies for binding to two gp41 constructs, one full-length MPER peptide, and two control peptides.

Figure 4. Comparison of wild-type and mutant MPER sequences.

Alignment of representative subject sequences containing the 4-residue MPER cassettes used in mutagenesis study aligned against the same subtype C consensus used in previous tables. The wild-type SVPC5 MPER sequence and the sequence of all three SVPC5-MPER mutants are also aligned. Residues 677, 680, 683, and 686 have been arbitrarily colored for easy identification. Residues in grey represent differences between the SVPC5 backbone and the envs described in the study. IC₅₀ and IC₉₀ values for mAb 4E10 are displayed for each Env.