Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8.

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.

Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox.

UNLABELLED: 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.

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.

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.

Potent neutralization of vaccinia virus by divergent murine antibodies targeting a common site of vulnerability in L1 protein.

UNLABELLED: 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.

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

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.

CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium.

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.

AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis.

Degeneration of photoreceptors is a common feature of ciliopathies, owing to the importance of the specialized ciliary structure of these cells. Mutations in AHI1, which encodes a cilium-localized protein, have been shown to cause a form of Joubert syndrome that is highly penetrant for retinal degeneration. We show that Ahi1-null mice fail to form retinal outer segments and have abnormal distribution of opsin throughout their photoreceptors. Apoptotic cell death of photoreceptors occurs rapidly between 2 and 4 weeks of age in these mice and is significantly (P = 0.00175 and 0.00613) delayed by a reduced dosage of opsin. This phenotype also shows dosage-sensitive genetic interactions with Nphp1, another ciliopathy-related gene. Although it is not a primary cause of retinal blindness in humans, we show that an allele of AHI1 is associated with a more than sevenfold increase in relative risk of retinal degeneration within a cohort of individuals with the hereditary kidney disease nephronophthisis. Our data support context-specific roles for AHI1 as a contributor to retinopathy and show that AHI1 may explain a proportion of the variability in retinal phenotypes observed in nephronophthisis.