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.

Distinct, IgG1-driven antibody response landscapes demarcate individuals with broadly HIV-1 neutralizing activity.

Understanding pathways that promote HIV-1 broadly neutralizing antibody (bnAb) induction is crucial to advance bnAb-based vaccines. We recently demarcated host, viral, and disease parameters associated with bnAb development in a large HIV-1 cohort screen. By establishing comprehensive antibody signatures based on IgG1, IgG2, and IgG3 activity to 13 HIV-1 antigens in 4,281 individuals in the same cohort, we now show that the same four parameters that are significantly linked with neutralization breadth, namely viral load, infection length, viral diversity, and ethnicity, also strongly influence HIV-1-binding antibody responses. However, the effects proved selective, shaping binding antibody responses in an antigen and IgG subclass-dependent manner. IgG response landscapes in bnAb inducers indicated a differentially regulated, IgG1-driven HIV-1 antigen response, and IgG1 binding of the BG505 SOSIP trimer proved the best predictor of HIV-1 neutralization breadth in plasma. Our findings emphasize the need to unravel immune modulators that underlie the differentially regulated IgG response in bnAb inducers to guide vaccine development.

Bacillus subtilis as a platform for molecular characterisation of regulatory mechanisms of Enterococcus faecalis resistance against cell wall antibiotics.

To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators.

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.

FixK2, a key regulator in Bradyrhizobium japonicum, is a substrate for the protease ClpAP in vitro.

FixK2 is a CRP-like transcription factor that controls the endosymbiotic lifestyle of Bradyrhizobium japonicum. The reason for its noticeable protease sensitivity was explored here. The repertoire of Clp chaperone-proteases in B. japonicum was examined, and specifically ClpAP1 and ClpXP1 were purified and tested. FixK2 was found to be degraded by ClpAP1 but not by ClpXP1. Degradation was inhibited by the ClpS1 adaptor protein, indicating that FixK2 is a direct substrate for ClpAP1. The last 12 amino acids of FixK2 appeared to be recognized by ClpA. The results suggest that the ClpAP system is involved in the cellular turnover of FixK2.