Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-MAb variants, VACV-66, VACV138, and VACV-304, separately bound to D8. While all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138 and VACV-304 binding sites along the CS binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high and low affinity CS binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer http://www.jbc.org/cgi/doi/10.1074/jbc.M117.814541 The latest version is at JBC Papers in Press. Published on November 9, 2017 as Manuscript M117.814541 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. at C A L FO R N IA IN ST IT U T E O F T E C H N O L O G Y on N ovem er 3, 2017 hp://w w w .jb.org/ D ow nladed from Human antibody responses to vaccinia virus D8 2 could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer. Smallpox is caused by infection with variola virus and was a major health threat until successful global vaccination efforts led to its complete eradication from the general human population (1). This eradication of an infectious agent was achieved by immunizing with Vaccinia virus (VACV), a related orthopoxvirus with low virulence and the active ingredient in the smallpox vaccine (2,3). Immunization with VACV leads to the robust production of highly neutralizing antibodies targeting several VACV envelope proteins, including A27, A33, B5, D8, H3, L1, and others, which are expressed on different viral envelopes (3,4). A27, D8, H3 and L1 are expressed on the outer membrane of the intracellular mature virion (IMV), while A33 and B5 are found in the more fragile extracellular enveloped virion (EEV), which has an additional host cell derived envelope. Both the IMV and the EEV particles are infectious virions, with IMV being more abundant and mainly responsible for spread between hosts, and EEV being involved in cell-to-cell spread within the host after infection with the IMV (5). As a result, a potent antibody-mediated immune response against VACV targets both infectious virions. Among the targeted envelope proteins, A27, D8, and H3 are glycosaminoglycan (GAG) adhesion molecules. GAGs are linear, mostly sulfated polysaccharides with repeating disaccharide units. The precise sulfation of each GAG is regulated by specific sulfotransferases, leading to the production of different isoforms. GAGs are covalently attached to various core proteins to form the proteoglycan (PG). They are ubiquitously expressed on cell surfaces and in extracellular matrices (6). In addition to providing structural integrity, GAGs participate in various physiological processes such as cell adhesion, cell proliferation, migration, neurite outgrowth, microbial adhesion and infection, inflammation and angiogenesis (7-11). Many pathogens encode GAG-binding proteins as a major route of entry (12). While A27 and H3 (13-15) bind to heparan sulfate (HS), D8 binds to chondroitin sulfate (CS) (16). Chondroitin sulfate (β-GlcA-β-(1-3)GalNAcsulfate) is the most abundant GAG and has 4 major isotypes (chondroitin-4-sulfate, CS-A; chondroitin6-sulfate, CS-C; chondroitin-2,6-sulfate, CS-D; chondroitin-4,6-disulfate, CS-E) in mammals (17). We have determined previously the structure of VACV D8 protein, which revealed an N-terminally located carbonic anhydrase (CAH) domain (residues 1-235), followed by a disordered stalk region (residues 236-267) that is required for higher oligomeric assembly of the D8 hexamer in solution, and for embedding D8 in the virial envelope (18,19). We have further identified CS-E as the optimal ligand for D8 and identified a positively charged crevice as the CS binding site (19). Computational docking suggested that the crevice is capable of binding to at least an octasaccharide of CS-E (18). We also previously characterized the binding pattern for a panel of D8-reactive murine antibodies, all of which targeted the CAH domain rather than the stalk region, and can be grouped into 4 different specificity groups based on crossblocking studies (18,20,21). One of these antibodies, LA5, is moderately neutralizing in the presence of complement and prevents D8 binding to CS-E, since LA5 binds above the CS-E binding crevice (18,19). However, to date, VACV epitopes that are targeted by human antibodies have not been characterized to atomic resolution using X-ray crystallography, and in total only the structures of eight murine antibodies bound to their VACV antigens A27, A33, D8, and L1 have been determined (19,22-25). Previously, we reported isolation of a panel of 21 anti-D8 human monoclonal antibodies (MAb) and assessed their neutralizing and protective capabilities (26). In this study, we have characterized the epitopes for some of these human D8 antibody and determined the first crystal structures of human antibodies bound to D8, using three clones designated VACV-66, VACV-138 and VACV-304. Similarly to the murine antibodies, human antibodies bound to the CAH domain of D8 and were moderately neutralizing in the presence of complement. Two antibodies, VACV-138 and VACV-304, also fully blocked D8 binding to CSA, while VACV-304 only partially blocked D8 binding to the high affinity ligand CS-E, revealing a highand low-affinity CS binding region within the central D8 crevice. RESULTS Human anti-D8 MAbs identify a novel D8 epitopeWe analyzed four human anti-D8 MAbs VACV-66, VACV-138, VACV-249, and VACV304 for their ability to block each other’s binding to recombinant D8 using a real-time binding assay (biolayer interferometry, BLI). VACV-66 was the at C A L FO R N IA IN ST IT U T E O F T E C H N O L O G Y on N ovem er 3, 2017 hp://w w w .jb.org/ D ow nladed from Human antibody responses to vaccinia virus D8 3 only antibody that did not compete with any other MAb for D8 binding, suggesting it bound a distinct epitope on D8 (Fig 1A). VACV-138, VACV-249, and VACV-304 all competed with each other for binding to D8, suggesting that their epitopes on D8 were overlapping. However, a previous study suggested that crossblocking of VACV-249 with VACV-304 depends on the order of antibodies used in the assay, suggesting the overlap is only partial (26). Next, we used ELISA to analyze the ability of VACV-66 and VACV-304, as representatives of the two major human MAb groups, to block binding of murine anti-D8 MAbs in a previously characterized antibody panel (21). We included representative murine MAbs from specificity groups I, II and IV to ask whether the human MAbs recognized common or unique epitopes on D8 (Fig 1B). While VACV-304 blocked binding of mouse CC7.1 (group II), human VACV-66 appeared to recognize a unique epitope on D8, notwithstanding that there was slight cross-blocking with the murine group IV antibodies HG12 and LA5 (Fig 1). LA5 had been shown previously to block D8 binding to its ligand CS-E (18,19), raising the possibility that some human MAbs may also prevent D8 binding to CS-E. Human and murine anti-D8 MAbs possess moderate neutralizing potencyWe next tested the ability of anti-D8 MAbs to neutralize IMV in a flow cytometry-based assay. While the murine control anti-L1 MAb M12B9 neutralized IMV in the absence of complement, most anti-D8 MAbs required the presence of complement to neutralize IMV, regardless of whether they were of human or murine origin (Fig 2). Murine MAb JF11 was the only anti-D8 MAb that did not neutralize, while neutralization mediated by MAb JE11 also was weak. Compared to L1, D8 appeared to be a target only for antibodies with a moderate level of neutralizing potency, likely because the GAG adhesion molecules A27 and H3 can compensate for the function of D8. Human anti-D8 MAbs bind with high affinityNext, we determined the binding affinity of human anti-D8 MAbs for recombinant D8. While VACV-66, VACV-138 and VACV-249 bound D8 with relatively low affinity (KD= 0.35 to 1.9 nM), VACV-304 bound with much higher affinity (KD=16pM, Fig 3). MAb VACV-249 had a comparably low association rate (kon= 2 x 10 1/Ms), while VACV-66, VACV-138 and VACV304 bound similarly fast (kon= 6.5 to 7.2 x 10 1/Ms). Surprisingly, the high affinity of VACV-304 was a result of extremely slow dissociation, which was roughly 100-times slower compared to VACV66 (koff= 1 x 10 vs. 1 x 10 1/s). This finding suggested that VACV-304 formed a stable complex with D8 that was characterized by a long half-life in solution. As the kinetic measurements were performed with immobilized intact IgG, only the 1:1 binding interaction between a single Fab and D8 was measured. Increases in avidity due to antibody bivalency were not addressed in this experimental design. As a result, binding of the antibody to D8 embedded

ABSTRACT Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen. IMPORTANCE Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an attempt to better understand the interplay between the antibodies and their antigens, we generated and functionally characterized a panel of anti-A27 antibodies and studied their interaction with the epitope using X-ray crystallography. We identified one protective antibody that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding. Analysis of the antibody-antigen interaction supports a model in which antibodies that can interfere with the functional activity of the antigen are more likely to confer protection than those that bind at the extremities of the antigen.

Meditope Biosciences’ SnAP (Site-specific novel Antibody Platform) technology provides a novel and specific way to create antibody drug conjugates (ADCs). Cyclic peptides, termed ‘meditopes’, have been designed to selectively bind to a ‘meditope binding site’ engineered into the Fab of an antibody. The process of conferring meditope binding is referred to as “meditope enablement”. Enabling antibodies for meditope binding does not interfere with antigen recognition or antibody integrity due to the unique location of the binding site. Meditope peptide binding can be utilized to attach cytotoxic payloads to an enabled antibody in a specific and consistent manner, and has the potential to dramatically improve the consistency of ADC production. For advanced hematological cancers such as CD33 positive acute myeloid leukemia (AML), using an anti-CD33 monoclonal antibody conjugated to a potent cytotoxic agent may be a useful tumor reduction strategy, especially when patients have failed other treatments. To this end, meditope enablement of lintuzumab and gentuzumab permits a consistent, site-specific attachment of cytotoxins with a highly controllable drug antibody ratio (DAR). Structural and biophysical characterization of the meditope enabled anti CD33 antibodies has provided key insight to the meditope enablement process. Experiments confirm that payload conjugation and meditope peptide binding does not interfere with antibody integrity or antigen recognition. The meditope peptide drug complexes were tested in cell-based assays for CD33-targeted cell killing. Using X-ray crystallographic analysis and guided docking, we propose modifications to further enhance affinity of the meditope peptide conjugate : CD33 antibody interaction. Citation Format: Calin D. Dumitru, Michael Matho, Elisabeth Gardiner, John C. Williams, Krzysztof Bzymek. Meditope enablement and structural analysis of anti-CD33 antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 869.

Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

Abstract Meditopes are cyclic peptides that bind the Fab fragment noncovalently, in a novel and unique site, outside the paratope. This property can be utilized to harness cytotoxic payloads to an antibody in a site-specific and consistent manner. One of the originally described peptides, named cQFD, presents a moderate affinity for the humanized antibody cetuximab (KD = 0.43 uM). The binding site involves residues from the heavy as well as the light chain of the antibody, and from both sides of the variable-to-constant hinge of the Fab domain, with a predominance for residues of the variable region. A similar affinity of cQFD to trastuzumab was achieved by mutating thirteen specific residues of the antibody, all located in the variable (Fv) region of the Fab domain; Nine of which are located in the light chain (LC: V9, I10, R39, T40, N41, G42, S43, R45, I83, D85, A100), two in the heavy chain (HC: S40 and I89). We refer to those mutations as to the “meditope enabling”. Via a combined approach, we (i) looked into peptide modifications to increase meditope's affinity for the antibody, and (ii) evaluated the transferability of this approach onto a panel of human antibodies of therapeutic relevance. Based on X-ray crystallography analysis, antibody modeling—in the case of antibodies for which no structure is available—and guided docking, we suggested a number of modifications (point mutations and/or peptide extension) that were subsequently tested via surface plasmon resonance. The top meditope leads were conjugated to MMAE and DM1 and their binding was reassessed to a number of humanized/IgG1 antibodies-confirming that payload conjugation does not interfere with meditope binding and also that meditope enabling and binding does not interfere with antigen binding. Finally, the complexes between antibodies and meditope-drug conjugates were tested in vitro in a panel of tumor cell lines, to measure and rank the efficacy of those leads in our ADC pipeline for further development as novel oncology therapeutics. Citation Format: Michael M. Matho, Elisabeth M. Gardiner, Calin D. Dumitru. Structurally guided development of meditope-based antibody-drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2015-646

ABSTRACT Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies. IMPORTANCE Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.

Mucosal dendritic cells (DCs) in the intestine acquire the unique capacity to produce retinoic acid (RA), a vitamin A metabolite that induces gut tropism and regulates the functional differentiation of the T cells they prime. Here, we identified a stromal cell (SC) population in the intestinal lamina propria (LP), which is capable of inducing RA production in DCs in a RA- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent fashion. Unlike DCs, LP SCs constitutively expressed the enzymatic machinery to produce RA even in the absence of dietary vitamin A, but were not able to do so in germ-free mice implying regulation by microbiota. Interestingly, DCs promoted GM-CSF production by the SCs indicating a two-way cross-talk between both cell types. Furthermore, RA-producing LP SCs and intestinal DCs localized closely in vivo suggesting that the interactions between both cell types might have an important role in the functional education of migratory DCs and therefore in the regulation of immune responses toward oral and commensal antigens.

The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4' and 6' of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

Abstract Immunization with vaccinia virus (VACV) confers cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibody (Ab) responses, however the specificity of human neutralizing antibodies to poxviruses is poorly understood. We used a highly-optimized human hybridoma technology to generate large panels of anti-VACV monoclonal Abs (mAbs) from vaccinia-immunized subjects or from subjects who had naturally acquired monkeypox infection. Immunologic characterization and DNA sequencing of the panel revealed 16 individual mAbs that recognized the immunodominant surface protein antigen D8. Several of anti-D8 mAbs possessed neutralizing activity against VACV, cowpox and monkeypox viruses. These Abs exhibited mainly a complement-dependent pattern of neutralization and they formed at least four cross-blocking groups, suggesting the existence of several non-overlapping neutralizing epitopes for D8. The results suggest that D8 is an important target for human neutralizing Abs generated following poxviral infection or vaccination, and suggest the existence of novel epitopes targeted by anti-D8 Abs that contribute to cross-protective immunity following smallpox vaccination.

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes.

Inbal Sela-Culang,1 Mohammed Rafii-El-Idrissi Benhnia,2,6,7 Michael H. Matho,3 Thomas Kaever,2 Matt Maybeno,2 Andrew Schlossman,2 Guy Nimrod,1 Sheng Li,4 Yan Xiang,5 Dirk Zajonc,3 Shane Crotty,2 Yanay Ofran,1,8,* and Bjoern Peters2,8,* 1The Goodman Faculty of Life Sciences, Nanotechnology Building, Bar Ilan University, Ramat Gan 52900, Israel 2Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA 3Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA 4Medicine and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92093, USA 5Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229, USA 6Present address: Department of Biochemistry and Molecular Biology and Immunology, University of Seville Medical School, 41009 Seville, Spain 7Present address: Laboratory of Immunovirology, Biomedicine Institute of Seville, Infectious Diseases Service, Virgen del Rocı́o University Hospital, 41013 Seville, Spain 8These authors contributed equally to this work *Correspondence: yanay@ofranlab.org (Y.O.), bpeters@liai.org (B.P.) http://dx.doi.org/10.1016/j.str.2014.02.003

ABSTRACT The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33.

Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Å(2) of protein surface.

IgE-mediated activation of mast cells and basophils underlies allergic diseases such as asthma. Histamine-releasing factor (HRF; also known as translationally controlled tumor protein [TCTP] and fortilin) has been implicated in late-phase allergic reactions (LPRs) and chronic allergic inflammation, but its functions during asthma are not well understood. Here, we identified a subset of IgE and IgG antibodies as HRF-interacting molecules in vitro. HRF was able to dimerize and bind to Igs via interactions of its N-terminal and internal regions with the Fab region of Igs. Therefore, HRF together with HRF-reactive IgE was able to activate mast cells in vitro. In mouse models of asthma and allergy, Ig-interacting HRF peptides that were shown to block HRF/Ig interactions in vitro inhibited IgE/HRF-induced mast cell activation and in vivo cutaneous anaphylaxis and airway inflammation. Intranasally administered HRF recruited inflammatory immune cells to the lung in naive mice in a mast cell- and Fc receptor-dependent manner. These results indicate that HRF has a proinflammatory role in asthma and skin immediate hypersensitivity, leading us to suggest HRF as a potential therapeutic target.

[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative mu-hydroxo Ni-Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro-peroxide.