Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

Abstract

Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.

Monoclonal Antibody 2C6 Targets a Cross-Clade Conformational Epitope in gp41 with Highly Active Antibody-Dependent Cell Cytotoxicity

“…Ab competition. In our previous studies, 2C6 interfered with biotinylated 8F6 binding, which targets the immunodominant loop of gp41, but the reciprocal was not shown (14). To identify the binding domain of 2C6 on gp41, a competition ELISA was performed using well-characterized anti-gp41 monoclonal Abs.…”

“…Peptide binding. We had previously utilized a group M consensus peptide set to attempt to map 2C6 binding to a linear epitope (14). We did not find a set of overlapping peptides that would be typical for a linear epitope targeting Ab.…”

“…3). The negative controls used in the figure were a gp41 C-terminal heptad repeat structure-targeting Ab 76-Q7-7C6 (14) and the CD4 binding site Ab VRC01. Notably, certain peptides in group M and MN sets showed nonspecific binding, so data are shown normalized to the internal negative-control Ab.…”

“…Despite the highly mutated nature of these Abs, which is often associated with neutralization for HIV-specific Abs, they are generally nnAbs. Previous competition studies using the most mutated (80 to 88% nucleotide homology to inferred germ line) of the Abs suggested targeting of three distinct epitopes and that these epitopes are conformationally dependent (14). Two of the structurally influenced epitopes were resolved, but the epitope bound by MAbs 76-Q3-2C6 and 76-Q3-6B8 (here termed 2C6 and 6B8, respectively) could not be resolved initially.…”

“…To enhance isolation of quaternary-targeting Abs (QtAbs), we previously used HIV-like particles (VLPs) to bind and sort individual B cells from long-term nonprogressor (LTNP) subjects to identify a panel of monoclonal Abs (MAbs) (14,15). When recombinant full-length MAbs were expressed using natively paired heavy-and light-chain sequences, a subset of these MAbs exhibited the binding profiles of QtAbs, preferably binding trimerized free and VLP-associated Env rather than monoclonal Env.…”