Elimination of HIV-1-infected cells by broadly neutralizing antibodies.

International audienceThe Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir

Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074–like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a “liganded” PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain

Human anti–HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness

The identification and characterization of conserved epitopes on the HIV-1 viral spike that are immunogenic in humans and targeted by neutralizing antibodies is an important step in vaccine design. Antibody cloning experiments revealed that 32% of all HIV-neutralizing antibodies expressed by the memory B cells in patients with high titers of broadly neutralizing antibodies recognize one or more “core” epitopes that were not defined. Here, we show that anti-core antibodies recognize a single conserved epitope on the gp120 subunit. Amino acids D474, M475, R476, which are essential for anti-core antibody binding, form an immunodominant triad at the outer domain/inner domain junction of gp120. The mutation of these residues to alanine impairs viral fusion and fitness. Thus, the core epitope, a frequent target of anti–HIV-neutralizing antibodies, including the broadly neutralizing antibody HJ16, is conserved and indispensible for viral infectivity. We conclude that the core epitope should be considered as a target for vaccine design

Broad Neutralization by a Combination of Antibodies Recognizing the CD4 Binding Site and a New Conformational Epitope on the HIV-1 Envelope Protein

Two to three years after infection, a fraction of HIV-1–infected individuals develop serologic activity that neutralizes most viral isolates. Broadly neutralizing antibodies that recognize the HIV-1 envelope protein have been isolated from these patients by single-cell sorting and by neutralization screens. Here, we report a new method for anti–HIV-1 antibody isolation based on capturing single B cells that recognize the HIV-1 envelope protein expressed on the surface of transfected cells. Although far less efficient than soluble protein baits, the cell-based capture method identified antibodies that bind to a new broadly neutralizing epitope in the vicinity of the V3 loop and the CD4-induced site (CD4i). The new epitope is expressed on the cell surface form of the HIV-1 spike, but not on soluble forms of the same envelope protein. Moreover, the new antibodies complement the neutralization spectrum of potent broadly neutralizing anti-CD4 binding site (CD4bs) antibodies obtained from the same individual. Thus, combinations of potent broadly neutralizing antibodies with complementary activity can account for the breadth and potency of naturally arising anti–HIV-1 serologic activity. Therefore, vaccines aimed at eliciting anti–HIV-1 serologic breadth and potency should not be limited to single epitopes

Somatic Mutations of the Immunoglobulin Framework Are Generally Required for Broad and Potent HIV-1 Neutralization

Broadly neutralizing antibodies (bNAbs) to HIV-1 can prevent infection and are therefore of great importance for HIV-1 vaccine design. Notably, bNAbs are highly somatically mutated and generated by a fraction of HIV-1-infected individuals several years after infection. Antibodies typically accumulate mutations in the complementarity determining region (CDR) loops, which usually contact the antigen. The CDR loops are scaffolded by canonical framework regions (FWRs) that are both resistant to and less tolerant of mutations. Here, we report that in contrast to most antibodies, including those with limited HIV-1 neutralizing activity, most bNAbs require somatic mutations in their FWRs. Structural and functional analyses reveal that somatic mutations in FWR residues enhance breadth and potency by providing increased flexibility and/or direct antigen contact. Thus, in bNAbs, FWRs play an essential role beyond scaffolding the CDR loops and their unusual contribution to potency and breadth should be considered in HIV-1 vaccine design

Adaptation of HIV-1 Envelope Glycoprotein gp120 to Humoral Immunity over the Course of the Epidemic

Since 2009, a large panel of broad and potent monoclonal neutralizing antibodies (MoNAbs) against HIV-1 have been isolated. These MoNAbs can protect from lllV-1 infection and suppress established infection in animal models. Because their efficacy should be evaluated in human clinical trials, it is of importance to define the sensitivity of the most contemporary transmitted variants to these MoNAbs. We, and others previously, reported that HIV-1 has become more resistant to neutralization over the course of the epidemic (Bunnik et al., Nature Med 2010, Bouvin-Pley et al., PloS Pathog 2013). Methods: Here we extended the analyses to the most potent MoNAbs described since then, either more recently isolated or improved by structure-based gene modifications. Results: We fully confirmed the first observations showing an increasing resistance of HIV-1 clade B over time to MoNAbs targeting the major gp l20 epitopes but not to MoNAbs targeting the gp41 MPER. Despite this evolution, some MoNAbs still were able to neutralize efficiently the most recently transmitted HIV-1 variants (2006-2010). The most potent MoNAbs were the bi-specific PG9- and PG16-iMab that alone were able to neutralize an variants at less than 0.4 mg/mL. The sensitivity to iMAb remained similar over time, suggesting that the trend of increasing resistance to PG9-/PG16-iMAb may be attributed only to die antigen binding domain of PG9/PG16. NIH45-46m2 (and -m7), 10-1074 and 10E8 were also highly potent and, if combined, reached the potency of PG9-/PG16-iMAb. We also observed that 3BNC 117 was almost as potent as the modified NIH45-46 antibodies, and that the lama-derived JM4IgG2b was the most potent Ab among those that do not target the major gp 120 neutralizing epitopes. Conclusions: These data clearly suggest a continuous drift of the env gene of HIV-1 elude B over the epidemic, and that not a single epitope is concerned but the entire gp120 as a whole. The consequences of this adaptation on the envelope functionality are being explored

Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding

Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals

Memory B Cell Antibodies to HIV-1 gp140 Cloned from Individuals Infected with Clade A and B Viruses

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency