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

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

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

Protection of Balb/c mice from VACV_WR by anti-A33 mAbs.

<p>Antibody protection against weight loss (A) and mortality (B) caused by VACV_WR challenge. All analyzed anti-A33 MAbs protect both against weight loss and death. One of two independent experiments is shown. Significance for all A33 antibodies (****) was P<0.0001, while the anti-L1 control antibody M12B9 was not significant (ns) with P = 0.1577. The negative control antibody A10 provided no protection as expected, while the positive control anti-B5 antibody B126 conferred protection. (B) Note that slight shifts on the Y-axis were implemented for visualization purposes for all antibodies that confer full protection.</p

Complement and isotype dependence of anti-A33 MAb neutralization of VACV EEV.

<p>VACV EEV neutralization activity of purified anti-A33 MAbs in the absence (MAbs) or presence (MAbs+10% C´) of complement. Rabbit anti-A33 polyclonal Abs (N628) were used as positive control. Anti-B5 MAbs B126 (IgG2a), and B96 (IgG1) were used as positive and negative neutralization controls, respectively. Human anti-ditrophenol (DNP, IgG1) and VACV EEV (EEV) were used as negative controls. Error bars indicate SEM in each condition. Dashed line indicates the 50% of plaques number of VACV EV in panels A and B. The data are representative of two experiments. Three more experiments were done and they show comparable results.</p

Data collection and refinement statistics of A33/Fab complexes.

<p>Data collection and refinement statistics of A33/Fab complexes.</p

Detailed contacts at the Ag:Fab interface.

<p>Interactions between A33 residues and light-chain (L, orange) or heavy chain (H, green) CDRs (1–3) of the MAbs A2C7, A20G2, and A27D7. Residues that elicit actual contacts as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005148#ppat.1005148.s003" target="_blank">S3 Table</a> are colored according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005148#ppat.1005148.g004" target="_blank">Fig 4</a>. Main chain is shown uniformly for residues eliciting VdW interactions, while individual atoms are colored for residues eliciting electrostatic interactions. Amino acid side chains are shown when relevant. A33 backbone in grey, and contact residues in black. Only main chain atoms are shown when residues are involved in VdW interactions.</p

Fab/A33 binding interactions.

<p>(A) Determination of A33/Fab binding stoichiometry by size exclusion chromatography (SEC). Fab/A33 complex formation of five different MAbs (A2C7, A26C7, A25D11, A27D7, and A20G2) is illustrated. Two major peaks corresponding in size to 1 Fab: 1 A33 dimer (red and blue curve) and 2 Fabs: 1 A33 dimer (brown, cyan and grey) complexes are visible. Presence of both Fab and A33 in each peak are confirmed by SDS-PAGE (bottom left corner). Molecular weight markers with sizes in kDa are shown as a reference (thin grey curve). (B) Real-time A33 binding curves to immobilized MAbs as assessed by BLI. Note that A33 dissociates much slower from MAbs A2C7 and A20G2, compared to A27D7, likely to its ability to simultaneous bind to two Fabs.</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 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