Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in Their Ability to Block D8 Binding to CS-E

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

Suppressor cell regulation of immune response to tumors: Abrogation by adult thymectomy

The regulatory role of the adult thymus on the appearance of cytotoxic and suppressor T cells (thymus-derived lymphocytes) to allogeneic and autochthonous virus-induced tumors in mice was investigated. It was demonstrated that C57BL/6 mice challenged with allogeneic P815 mastocytoma cells and complete Freund's adjuvant failed to develop cytotoxic cells but instead developed suppressor T cells which inhibited cytotoxic T cell function. Further, adjuvant-induced suppressor cells prevented the primary in vitro induction of cytotoxic T cells to P815 mastocytoma cells. In contrast, adult thymectomized animals, when challenged with adjuvant and allogeneic cells, had a normal cytotoxic response in vivo and their cells could not inhibit the generation of cytotoxic T cells in vitro. These studies suggested that the intact adult thymus was necessary for the induction of suppressor cells. Moreover, suppressor cells regulated cytotoxic T cell activity both in vivo and in vitro. Further, it was shown that adjuvant could prevent the normal immune response to virus-induced tumors. BALB/c mice treated with murine sarcoma virus developed tumors which reached a maximal size by day 14 and then regressed. Sham thymectomized animals treated with virus and complete Freund's adjuvant to generate suppressor cells died from progressive tumor growth. In contrast, thymectomized animals similarly treated had normal regression of tumor and survived. These studies lead to the conclusion that the adult thymus may regulate immune responsiveness by the export of suppressor T cells which regulate other T cell responses to both allogeneic and tumor antigens

Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

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

Group I (JE11) footprint.

<p><b>A.</b> EM reconstruction of the D8 monomer in complex with Fab's JE11 (group I) and LA5 (group IV) at 24 Å resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495.s005" target="_blank">figure S5</a>. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495-SelaCulang1" target="_blank">[9]</a>: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. <b>B.</b> Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495-SelaCulang1" target="_blank">[9]</a>. <b>C.</b> Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding.</p

D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E.

<p><b>A.</b> GAG microarray performed with monomeric D8 antigen. <b>B.</b> MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. <b>C.</b> MAb/CS-E cross-blocking of group III MAbs <b>D.</b> Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).</p

Group III (EE11) footprint.

<p><b>A.</b> EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495.s007" target="_blank">figure S7</a>. Epitope footprints follow the same color code as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495-SelaCulang1" target="_blank">[9]</a>: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. <b>B.</b> Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. <b>C.</b> Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes).</p

D8 oligomeric interface is secluded to the C-terminal region 235–262.

<p><b>A.</b> From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). <b>B.</b> SEC profiles of recombinant D8 Δ235, Δ262, and Δ263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. <b>C.</b> Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. <b>D.</b> CS-E microarray indicating that D8 hexamer (0.33 µM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 µM) <b>E.</b> GAG microarray obtained with D8 oligomer (0.1 µM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).</p

Group II (CC7.1) footprint.

<p><b>A.</b> EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and CC7.1 (group II) at 21 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495.s006" target="_blank">figure S6</a>. Epitope footprints follow the same color code as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004495#ppat.1004495-SelaCulang1" target="_blank">[9]</a>: group I (JE11): red; group II (CC7.1): green. <b>B.</b> Summary of CC7.1 (group II) contacts. D8 residues colored in forest green belong to the initial definition of group I epitope, assessed by alanine scanning. Lighter green residues complete the definition of group II epitope. <b>C.</b> Footprint of completed CC7.1 epitope. Forest green and light green footprints evidence initial and current epitope definitions. Group IV (LA5) footprint in orange does not intersect with group II (CC7.1) epitope.</p

Mapping of the CS-E binding site on vaccinia D8 ectodomain.

<p><b>A.</b> Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. <b>B.</b> Mapping of CS-E binding site. Mutation R220A led to a ∼50% decrease in CS-E binding compared to wt, while CS-E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments.</p