Rabbit anti-HIV-1 monoclonal antibodies raised by immunization can mimic the antigen-binding modes of antibodies derived from HIV-1-infected humans

The rabbit is a commonly used animal model in studying antibody responses in HIV/AIDS vaccine development. However, no rabbit monoclonal antibodies (MAbs) have been developed previously to study the epitope-specific antibody responses against HIV-1 envelope (Env) glycoproteins, and little is known about how the rabbit immune system can mimic the human immune system in eliciting such antibodies. Here we present structural analyses of two rabbit MAbs, R56 and R20, against the third variable region (V3) of HIV-1 gp120. R56 recognizes the well-studied immunogenic region in the V3 crown, while R20 targets a less-studied region at the C terminus of V3. By comparison of the Fab/epitope complex structures of these two antibodies raised by immunization with that of the corresponding human antibodies derived from patients chronically infected with HIV-1, we found that rabbit antibodies can recognize immunogenic regions of gp120 and mimic the binding modes of human antibodies. This result can provide new insight into the use of the rabbit as an animal model in AIDS vaccine development

Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the beta-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope. IMPORTANCE: The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env

Automatic detection of cone photoreceptors with fully convolutional networks

Purpose: To develop a fully automatic method, based on deep learning algorithms, for determining the locations of cone photoreceptors within adaptive optics scanning laser ophthalmoscope images and evaluate its performance against a dataset of manually segmented images. Methods: A fully convolutional network (FCN) based on U-Net architecture was used to generate prediction probability maps and then used a localization algorithm to reduce the prediction map to a collection of points. The proposed method was trained and tested on two publicly available datasets of different imaging modalities, with Dice overlap, false discovery rate, and true positive reported to assess performance. Results: The proposed method achieves a Dice coefficient of 0.989, true positive rate of 0.987, and false discovery rate of 0.009 on the first confocal dataset; and a Dice coefficient of 0.926, true positive rate of 0.909, and false discovery rate of 0.051 on the second split detector dataset. Results compare favorably with a previously proposed method, but this method provides quicker (25 times faster) evaluation performance. Conclusions: The proposed FCN-based method demonstrates that deep learning algorithms can achieve accurate cone localizations, almost comparable to a human expert, while labeling the images. Translational Relevance: Manual cone photoreceptor identification is a time-consuming task due to the large number of cones present within a single image; using the proposed FCN-based method could support the image analysis task, drastically reducing the need for manual assessment of the photoreceptor mosaic.</p

Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors▿

Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation

Structure-Based Functional Characterization of Repressor of Toxin (Rot), a Central Regulator of Staphylococcus aureus Virulence

Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus

Structure-Based Functional Characterization of Repressor of Toxin (Rot), a Central Regulator of Staphylococcus aureus Virulence

Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus

Crystal Structure of a Preacylation Complex of the β‑Lactamase Inhibitor Sulbactam Bound to a Sulfenamide Bond-Containing Thiol-β-lactamase

The rise of inhibitor-resistant and other β-lactamase variants is generating an interest in developing new β-lactamase inhibitors to complement currently available antibiotics. To gain insight into the chemistry of inhibitor recognition, we determined the crystal structure of the inhibitor preacylation complex of sulbactam, a clinical β-lactamase inhibitor, bound in the active site of the S70C variant of SHV-1 β-lactamase, a resistance enzyme that is normally present in <i>Klebsiella pneumoniae</i>. The S70C mutation was designed to affect the reactivity of that catalytic residue to allow for capture of the preacylation complex. Unexpectedly, the 1.45 Å resolution inhibitor complex structure revealed that residue C70 is involved in a sulfenamide bond with K73. Such a covalent bond is not present in the wild-type SHV-1 or in an apo S70C structure also determined in this study. This bond likely contributed significantly to obtaining the preacylation complex with sulbactam due to further decreased reactivity toward substrates. The intact sulbactam is positioned in the active site such that its carboxyl moiety interacts with R244, S130, and T235 and its carbonyl moiety is situated in the oxyanion hole. To our knowledge, in addition to being the first preacylation inhibitor β-lactamase complex, this is also the first observation of a sulfenamide bond between a cysteine and lysine in an active site. Not only could our results aid, therefore, structure-based inhibitor design efforts in class A β-lactamases, but the sulfenamide-bond forming approach to yield preacylation complexes could also be applied to other classes of β-lactamases and penicillin-binding proteins with the SXXK motif

Crystal Structure of a Preacylation Complex of the β‑Lactamase Inhibitor Sulbactam Bound to a Sulfenamide Bond-Containing Thiol-β-lactamase

The rise of inhibitor-resistant and other β-lactamase variants is generating an interest in developing new β-lactamase inhibitors to complement currently available antibiotics. To gain insight into the chemistry of inhibitor recognition, we determined the crystal structure of the inhibitor preacylation complex of sulbactam, a clinical β-lactamase inhibitor, bound in the active site of the S70C variant of SHV-1 β-lactamase, a resistance enzyme that is normally present in <i>Klebsiella pneumoniae</i>. The S70C mutation was designed to affect the reactivity of that catalytic residue to allow for capture of the preacylation complex. Unexpectedly, the 1.45 Å resolution inhibitor complex structure revealed that residue C70 is involved in a sulfenamide bond with K73. Such a covalent bond is not present in the wild-type SHV-1 or in an apo S70C structure also determined in this study. This bond likely contributed significantly to obtaining the preacylation complex with sulbactam due to further decreased reactivity toward substrates. The intact sulbactam is positioned in the active site such that its carboxyl moiety interacts with R244, S130, and T235 and its carbonyl moiety is situated in the oxyanion hole. To our knowledge, in addition to being the first preacylation inhibitor β-lactamase complex, this is also the first observation of a sulfenamide bond between a cysteine and lysine in an active site. Not only could our results aid, therefore, structure-based inhibitor design efforts in class A β-lactamases, but the sulfenamide-bond forming approach to yield preacylation complexes could also be applied to other classes of β-lactamases and penicillin-binding proteins with the SXXK motif