Epitopes for broad and potent neutralizing antibody responses during chronic infection with human immunodeficiency virus type 1

Abstract

Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved nonlinear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection.

Figure 1

Use of the glycan-dependent epitopes on the gp120 outer domain. To quantify and compare the neutralization sensitivity of the wild-type and N332S HIV-1_JR-FL gp160s to mAb 2G12 and subject antisera, the residual infectivity was measured using the single-round entry/neutralization assay. The luciferase activity of the mock-treated virus was used as the index of total infectivity, set as 100%, to which the infectivity of antibody-treated viruses was standardized as a percentage. The wild-type and N332S mutant Envs were neutralized by mAb 2G12 (A), antiserum C1-343 (B), and antiserum C1-259 (C). The same molar amounts of gp140 trimer (40 ug), gp120 monomer (13.3 ug), and OD1 (5 ug) were used to deplete neutralizing activity from antisera C1-343 (D) and C1-259 (E). The same amount of bare Ni-NTA Superflow beads (Qiagen), designated as “No Nickel”, was used as negative control.

Figure 2

Use of other neutralizing epitopes on the gp120 outer domain. A. 5 ug of either OD1 or OD1ΔV3 proteins were used to deplete nAbs from subject antisera. Due to variation in different experimental sessions, the data on UAB-D represent averages of 4 independent experiments. B. The neutralizing activity that survived the competition with 10 ug/ml of the HIV-1_JR-FL V3 peptide was measured using the standard neutralization assay, compared with same antiserum mock-treated with culture medium. The same quantity of peptide was also used to treat the target virus in parallel, and demonstrated no detectable inhibition of virus infectivity (data not shown).

Figure 3

Serum neutralizing activity from subject C1-328 is sensitive to a change in the CD4BS region. Luciferase reporter viruses carrying the wild-type or T257A HIV-1_JR-FL gp160s were subjected to neutralization by b12, (A) or antiserum C1-328 (B).

Figure 4

Detection of CD4BS nAbs using depletion by recombinant Env proteins. A. The wild-type and D368R mutant HIV-1_JR-FL gp120 monomer and the gp140(−/FT) trimer were expressed in 293F cells and purified by Ni-NTA affinity column. Ten ug of the final products were analyzed on an 8% SDS-PAGE gel under reducing and non-reducing conditions and visualized by Coomassie blue staining. B. Same molar amounts of the wild-type or D368R gp120 monomer, or the gp140 trimer were used to deplete neutralizing activity from subject antisera. The neutralizing activity in the final flow-through was measured using the standard neutralization assay, in parallel to the same antiserum mock-depleted by empty Ni-NTA beads. Due to variations in different experimental sessions, the data on UAB-D represent averages of 4 independent experiments.