Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

Conformation-dependent recognition of HIV gp120 by designed ankyrin repeat proteins provides access to novel HIV entry inhibitors.

Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

MPER-specific antibodies induce gp120 shedding and irreversibly neutralize HIV-1.

Interference with virus entry is known to be the principle mechanism of HIV neutralization by antibodies, including 2F5 and 4E10, which bind to the membrane-proximal external region (MPER) of the gp41 envelope protein. However, to date, the precise molecular events underlying neutralization by MPER-specific antibodies remain incompletely understood. In this study, we investigated the capacity of these antibodies to irrevocably sterilize HIV virions. Long-term effects of antibodies on virions can differ, rendering neutralization either reversible or irreversible. MPER-specific antibodies irreversibly neutralize virions, and this capacity is associated with induction of gp120 shedding. Both processes have similar thermodynamic properties and slow kinetics requiring several hours. Antibodies directed to the CD4 binding site, V3 loop, and the MPER can induce gp120 shedding, and shedding activity is detected with high frequency in plasma from patients infected with divergent genetic HIV-1 subtypes. Importantly, as we show in this study, induction of gp120 shedding is closely associated with MPER antibody inhibition, constituting either a primary event leading to virion neutralization or representing an immediate consequence thereof, and thus needs to be factored into the mechanistic processes underlying their activity.

HIV sensitivity to neutralization is determined by target and virus producer cell properties.

OBJECTIVE: Using clinical isolates from a recent passive immunization trial with antibody 2G12, we probed the capacity of frequently used neutralization formats - the primary peripheral blood mononuclear cell (PBMC)-based and the TZM-bl cell-based assay systems - to predict in-vivo activity of 2G12. DESIGN: Antibodies and entry inhibitors of established efficacy were used to neutralize HIV-1 isolates in different in-vitro assay setups. METHODS: Single round infection with Env-pseudotyped and multiple round infection with replication-competent virus was studied on PBMCs and a variety of engineered cell lines. RESULTS: Six out of 12 isolates with high sensitivity to 2G12 in the replication-competent PBMC assay lacked sensitivity to the monoclonal antibody in the env-pseudotype TZM-bl assay. Outcome of passive immunization with 2G12 corroborated the PBMC-assay in-vitro data, as escape mutations to 2G12 emerged, proving the monoclonal antibody's impact on HIV in vivo. Failure to inhibit pseudotype infection of TZM-bl was not due to sequence differences or the pseudotype infection per se, as infection of PBMCs with the identical pseudotyped viruses was sensitive to 2G12 inhibition. Similar shifts in efficacy, though less extreme, were noted for other neutralizing antibodies and inhibitors. Exploration of causes for the observed differences between assay systems revealed that both target cell and virus producer properties influence sensitivity of virus entry to inhibition. CONCLUSION: Our observation that neutralization assay systems employing engineered reporter cell lines can miss in-vivo relevant neutralizing activities strongly argues that preclinical assessment should not be restricted to a single assay type.

Divergent effects of cell environment on HIV entry inhibitor activity.

OBJECTIVE: Successful HIV vaccine and entry inhibitor development depends on use of assay systems that closely reflect in-vivo activities. Recent reports suggest that the currently most widely used assay format, which relies on the genetically engineered target cell line TZM-bl, can fail to detect certain neutralization activities detected on primary peripheral blood mononuclear cell (PBMC)-based assay systems. In the present study, we investigate the influence the target cell context bears on HIV entry inhibition. DESIGN: In a comprehensive survey, the effect of 11 neutralizing antibodies and inhibitors in blocking entry of 30 envelope pseudotyped virus strains in two types of target cells, PBMC and TZM-bl, was evaluated. METHODS: Env-pseudotyped HIV infection of PBMC and TZM-bl cells. RESULTS: We demonstrate here that depending on the type of inhibitor, relative neutralization potencies are shifted to a variable extent and direction on TZM-bl and PBMC cells. In our assay set up, differences in inhibitor activity were solely effected by the target cell environment and amounted up to 2-3 logs lower activity on TZM-bl cells in several cases. Overall, neutralizing antibodies, 2G12, 2F5 and 4E10, were less active in the TZM-bl system, whereas CD4 binding site directed inhibitor activities were detected equally well on both target cells, raising concerns that the TZM-bl assay may overrate the relevance of CD4 binding site specific responses. CONCLUSION: Our data strongly argue that preclinical assessment should not be restricted to a single type of assay, as systematic underestimation or overestimation of activities would be inevitable.

CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.