Jared M. Sampson

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.

Human Anti-V3 HIV-1 Monoclonal Antibodies Encoded by the VH5-51/VL Lambda Genes Define a Conserved Antigenic Structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

Rabbit Anti-HIV-1 Monoclonal Antibodies Raised by Immunization Can Mimic the Antigen-Binding Modes of Antibodies Derived from HIV-1-Infected Humans.

ABSTRACT 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.

Functional Implications of the Binding Mode of a Human Conformation-dependent V2 Monoclonal Antibody against HIV

ABSTRACT Data from the RV144 HIV vaccine trial indicated that gp120 V2 antibodies were associated with a lower risk of infection; thus, the mapping of V2 epitopes can contribute to the design of an effective HIV vaccine. We solved the crystal structure of human monoclonal antibody (MAb) 2158, which targets a conformational V2 epitope overlapping the α4β7 integrin binding site, and constructed a full-length model of V1V2. Comparison of computational energy stability to experimental enzyme-linked immunosorbent assay (ELISA) results identified a hydrophobic core that stabilizes the V2 region for optimal 2158 binding, as well as residues that directly mediate side chain interactions with MAb 2158. These data define the binding surface recognized by MAb 2158 and offer a structural explanation for why a mismatched mutation at position 181 (I181X) in the V2 loop was associated with a higher vaccine efficiency in the RV144 clinical vaccine trial. IMPORTANCE Correlate analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the second variable region (V2) of HIV-1 gp120 was responsible for the modest protection observed in the trial. V2 is a highly variable and immunogenic region, and structural information on its antigenic landscape will be important for rational design of an effective HIV-1 vaccine. Using X-ray crystallography, computational design tools, and mutagenesis assays, we carried out a detailed and systematic investigation of the epitope recognition of human V2 MAb 2158 and demonstrated that its epitope region overlaps the integrin binding site within V2. In addition, we propose a structure-based mechanism for mismatching of the isoleucine at position 181 and the increased vaccine efficacy seen in the RV144 vaccine trial.

Structural Analysis of Human and Macaque mAbs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 Å in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

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

ABSTRACT 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 β-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.

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 Klebsiella pneumoniae. 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.

Limonene reduces hyperalgesia induced by gp120 and cytokines by modulation of IL-1 β and protein expression in spinal cord of mice

Aims: We have investigated the antihyperalgesic effects of limonene in mice that received intrathecal injection of gp120. Main methods: Male Swiss mice received gp120, IL‐1&bgr; or TNF‐&agr; intrathecally or sterile saline as a control. A mechanical sensitivity test was performed at 2 and 3 h after the injection. Spinal cord and blood samples were isolated for protein quantification. Key findings: Intrathecal administration of gp120 increased mechanical sensitivity measured with an electronic Von Frey apparatus, at 2 and 3 h after the injections. Limonene administered orally prior to gp120 administration significantly decreased this mechanical sensitivity at 3 h after the gp120 injection. In addition, intrathecal injection of gp120 increased IL‐1&bgr; and IL‐10 in serum, and limonene prevented the ability of gp120 to increase these cytokines. Limonene also inhibited TNF‐&agr; and IL‐1&bgr;‐induced mechanical hyperalgesia. Western blot assay demonstrated limonene was capable of increasing SOD expression in the cytoplasm of cells from spinal cord at 4 h after intrathecal IL‐1&bgr; injection. Significance: These results demonstrate that gp120 causes mechanical hyperalgesia and a peripheral increase in IL‐1&bgr; and IL‐10, and that prior administration of limonene inhibits these changes. Also limonene modulates the activation of SOD expression in the spinal cord after spinal IL‐1&bgr; application. The ability of limonene to inhibit the mechanical hyperalgesia induced by gp120, TNF‐&agr; and IL‐1&bgr; emphasizes the anti‐inflammatory action of limonene, specifically its ability to inhibit cytokine production and its consequences.

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 analysis of anti-V2 mAb 2158 suggests a conformational epitope involving an N-linked glycan.

Background Many structural elements of HIV-1 gp120 have been revealed by X-ray crystallography, NMR and electron microscopy; however, several key immunogenic regions are still not well understood. Data from the recent RV144 trial indicated that antibodies targeting the V1V2 region correlate with a lower risk of infection. A detailed characterization of anti-V2 antibodies, in concert with recent V1V2 structural information is critical to the design of a V2 immunogen.