Biology subjects

Vance, D. J.

Publications and source records attributed to Vance, D. J..

10 recordsLinked to original sources

bioRxiv2025

A Broadly Conserved Protective Epitope on the Lyme Disease Vaccine Antigen, OspA

Lyme disease, caused by the spirochete, Borreliella burgdorferi sensu latu (Bbsl), is a debilitating tickborne infection of increasing incidence in North America, Europe and Asia. While vaccines based on Outer surface protein A (OspA) have proven highly efficacious at blocking Bbsl tick-to-human transmission, the high degree of antigenic variability among the major OspA serotypes (ST) has made the development of a broadly cross protective vaccine difficult. Recent profiling of protective human monoclonal antibodies (mAbs) has suggested the existence of conserved epitopes situated within OspAs central {beta}-sheet (CBS), although direct comparisons of cross-serotype functionality has been hindered by biological differences among the major Bbsl genospecies. To address these issues, we developed a panel of isogenic B. burgdorferi viability reporter strains expressing the seven major OspA serotypes (ST1-7) and probed them with CBS-targeting mAbs to evaluate their complement-dependent borreliacidal activity. The mAbs segregated into three distinct classes with varying degrees of borreliacidal activity: class 1 mAbs exhibited potent killing against all seven OspA serotypes, while classes 2 and 3 had restricted or no activity against two of the seven serotypes. Structural analysis of Fabs derived from each class of mAbs in complex with OspA ST1 showed that they target overlapping epitopes spanning {beta}-strands 6-10 and involve contact with largely invariant residues. Further analysis of B. burgdorferi reporter strains expressing OspA variants from 17 additional Bbsl genospecies identified Lys-107 as a determinant of susceptibility for nearly all CBS mAbs. Taken together, these findings raise the prospect of structure-based design of a broadly protective monovalent Lyme disease vaccine. AUTHOR SUMMARYLyme disease, caused by the spirochete, Borreliella burgdorferi sensu latu (Bbsl), is a potentially debilitating tickborne infection of increasing incidence in North America, Europe and Asia. While vaccines based on Outer surface protein A (OspA) have proven highly efficacious at blocking Bbsl tick-to-human transmission, the genetic and serological heterogeneity of OspA across Borrelia genospecies has complicated matters. In this report, we use a collection of transmission-blocking human monoclonal antibodies to delineate a protective region (epitope) within the central core of OspA that is conserved across all major OspA serotypes. These results have important implications for engineering a broadly reactive Lyme disease vaccine.

immunology↗

bioRxiv2025

An Fc-silent OspA monoclonal antibody passively protects mice from tick and intradermal Borrelia burgdorferi challenge

The monoclonal antibody, LA-2, has played a pivotal role in the development of Outer surface protein A (OspA)-based vaccines for Lyme disease, a multisystem illness caused by the tick-borne spirochete, Borrelia burgdorferi sensu lato. Of particular significance was the demonstration more than three decades ago that LA-2 equivalent antibody titers, defined by a competitive-inhibition ELISA, serve as a reliable correlate of vaccine-induced protection across different species, including humans. In vitro characterization of LA-2 has identified both complement-dependent and -independent activities, although which of these attributes contribute to protection against B. burgdorferi remains unresolved. To address this issue, we generated and characterized an "Fc-silent" version of LA-2 IgG1 carrying so-called LALAPG substitutions (L234A, L235A, P329G). We demonstrate that LA-2 LALAPG retained OspA binding activity but was severely attenuated in in vitro complement deposition and complement-dependent borreliacidal assays. Nonetheless, LA-2 LALAPG was as effective as LA-2 at passively protecting C3H mice against nymphal tick-mediated B. burgdorferi challenge. LA-2 LALAPG was also equivalent to LA-2 in passively protecting BALB/c mice against intradermal B. burgdorferi challenge. In the intradermal challenge model, viable spirochetes were not recoverable 24 h after injection from skin biopsies of mice treated with LA-2 or LA-2 LALAPG, and an influx of pro-inflammatory cytokines and chemokines to the injection site was abrogated. Collectively, these results suggest that LA-2s primary mode of action involves direct physical interactions with the spirochete rather than complement-dependent killing. Elucidating these mechanisms may have implications for understanding the mechanistic correlates of OspA-based vaccine-induced immunity in humans.

immunology↗

bioRxiv2024

A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication

Expression of camelid-derived, single-domain antibodies (VHHs) within the cytoplasm of mammalian cells as "intrabodies" has opened-up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein (RIP). The intrabody consists of two structurally defined VHHs that target distinct epitopes on RTs enzymatic subunit (RTA): V9E1 targets RTAs P-stalk recruitment site, and V2A11 targets RTAs active site. Resistance to RT conferred by the biparatopic VHH construct far exceeded that of either of the VHHs alone and effectively inhibited all measurable RT-induced cytotoxicty in vitro. We propose that targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.

immunology↗

bioRxiv2024

A refined human linear B cell epitope map of Outer surface protein C (OspC) from the Lyme disease spirochete, Borreliella burgdorferi

A detailed understanding of the human antibody response to Outer surface protein C (OspC) of Borrelellia burgdorferi has important implications for Lyme disease diagnostics and vaccines. In this report, a total of 13 peptides encompassing eight reported OspC linear B cell epitopes from OspC types A, B and K, including the conserved C-terminus (residues 193-210: peptide C10), were evaluated by multiplex immunoassay (MIA) for IgG reactivity with [~]700 human serum samples confirmed positive in a two-tiered Lyme disease diagnostic assay and [~]160 post-treatment Lyme disease (PTLD) serum samples. The VlsE C6-17 peptide was included as a positive control. Diagnostic serum IgG reacted with 11 of the 13 OspC-derived peptides, significantly more than controls, with the C10 peptide being the most reactive. In the PTLD serum samples, two OspC peptides including C10 were significantly more reactive than controls. Spearmans rank correlation matrices and hierarchical clustering indicated a strong correlation between C10 and VlsE C6-17 peptide reactivity but little demonstrable association between C10 and the other OspC peptides or recombinant OspC. OspC peptide reactivities (excluding C10) were strongly correlated with each other and were disproportionately influenced by a subset of pan-reactive samples. In the PTLD cohort, C10 clustered with the other OspC-derived peptides and was distinct from OspC and VlsE C6-17. The asynchronous serologic response to OspC, C10, and the OspC-derived peptides reveals the complexity of B cell responses to B. burgdorferi and confounds simple interpretation of antibody profiles associated with Lyme disease. IMPORTANCELyme disease is an emerging tick-borne infection caused by the spirochete, Borreliella burgdorferi. In humans, antibodies against spirochetal outer surface lipoproteins are proposed to play a role in disease resolution and in protection against reinfection. Some of those same antibodies also serve as diagnostic indicators of an active or history of Lyme disease. In this study, we sought to validate reported antibody binding sites on Outer surface protein C (OspC), a known target of both protective and diagnostic antibodies.

immunology↗

bioRxiv2024

Single-domain antibodies reveal unique borreliacidal epitopes on the Lyme disease vaccine antigen, Outer surface protein A (OspA)

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, Outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 (ST1) from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK(R) Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition ELISAs with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry (HX-MS). Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being conserved epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

immunology↗

bioRxiv2022

Structural Elucidation of a Protective B cell Epitope on Outer Surface Protein C (OspC) of the Lyme disease spirochete, Borreliella burgdorferi

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5s epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspCs -helix 1 and -helix 6, as well as interactions with the loop between a-helices 5 and 6. In addition, B5s complementarity-determining region (CDR) H3 bridged the OspC-OspC homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease. IMPORTANCEThe spirochete, Borreliella burgdorferi, is the causative agent of Lyme borreliosis, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal. After B. burgdorferi is deposited into the skin of a human host, it replicates locally and spreads systemically, often resulting in clinical manifestations involving the central nervous system, joints, and/or heart. Antibodies directed against B. burgdorferis outer surface protein C (OspC) are known to block tick-to-host transmission, as well as dissemination of the spirochete within a mammalian host. In this report, we reveal the first atomic structure of one such antibody in complex with OspC. Our results have implications for the design of a Lyme disease vaccine capable to interfering with multiple stages in B. burgdorferi infection.

immunology↗

bioRxiv2022

The Flexible Linker of Decorin Binding Protein A from Borrelia burgdorferi is a Target of Antibodies in Lyme disease

Decorin binding protein A (DbpA) is a surface adhesin expressed by Borrelia burgdorferi, the causative agent of Lyme disease. While DbpA is one of the most immunogenic B. burgdorferi antigens in Lyme disease patients, the B cell epitopes recognized over the course of infection have not been defined. In this report we profiled [~]300 human serum samples from early, mid, and late-stage Lyme disease for IgM and IgG reactivity with DbpA and a tiled DbpA 18-mer peptide array derived from B.b. strains B31 and 297. Using ELISA and multiplex immunoassays (MIA), we identified 12 DbpA-derived peptides whose reactivity was elevated in Lyme disease patients as compared to healthy controls. The most reactive peptide ("A7") corresponds to the flexible loop between DbpAB31 -helix 1 and -helix 2, implicated in influencing DbpA binding to heparin and dermatan sulfate. An A7-like peptide is also reportedly a target of antibodies in Lyme neuroborreliosis patients. The remaining peptides, while highly reactive in serum samples across disease stages, likely represent non-native epitopes, as antibody reactivity to a subset of peptides in competition assays was unaffected by the addition of soluble DbpA. Moreover, peptide reactivity of any given patient sample rarely correlated with overall DbpA antibody levels, suggesting the antibodies were raised against DbpA degradation products. Insights into B cell epitopes on DbpA elicited during Lyme disease have important implications for understanding how B.burgdorferi persists in the face of an overwhelming antibody response. IMPORTANCEThe bacterium, Borrelia burgdorferi, is the causative agent of Lyme disease, the most reported tick-borne illness in the United States. In humans, clinical manifestations of Lyme disease are complex and can persist for months, even in the face of a robust antibody response directed against numerous B. burgdorferi surface proteins, including decorin binding protein A (DbpA), which is involved in early stages of infection. In this study we employed [~]300 serum samples from Lyme disease patients to better understand antibody reactivity with specific regions ("epitopes") of DbpA. We identified one epitope on DbpA recognized by antibodies from almost half of the serum samples tested and that, theoretically, should block DbpAs ability to function in promoting B. burgdorferi colonization of human hosts.

immunology↗

bioRxiv2020

Sites of Vulnerability on Ricin B Chain Revealed through Epitope Mapping of Toxin-Neutralizing Monoclonal Antibodies

Ricin toxins B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes efficient uptake and intracellular trafficking of ricins ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (, {beta}, {gamma}). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1 and 2{gamma}) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit ([~]90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTBs duplicative structure. To circumvent this problem, full-length RTB or the two individual domains, RTB-D1 and RTB-D2, were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. The results indicated that SylH3 captured RTB-D1, while 24B11 captured RTB-D2. Analysis of additional toxin-neutralizing and non-neutralizing mAbs along with single chain antibodies (VHHs) known to compete with SylH3 or 24B11 confirmed these domain assignments. These results not only indicate that so-called type I and type II mAbs segregate on the basis of domain specificity, but suggest that RTBs two domains may contribute to distinct steps in the intoxication pathway.

immunology↗

bioRxiv2019

Intracellular Neutralization of Ricin Toxin by Single Domain Antibodies Targeting the Active Site Pocket

The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (VHHs) previously reported to recognize epitopes in proximity to RTAs active site. Three of the VHHs, V2A11, V8E6 and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies (\"intrabodies\"). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTAs active site and interact with key catalytic residues. V2G10, in contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four VHHs also penetrated/occluded RTAs active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.

immunology↗

bioRxiv2018

Fine Specificity Epitope Analysis by HX-MS Identifies Contact Points on Ricin Toxin Recognized by Protective Monoclonal Antibodies

Ricin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report we describe five new mouse monoclonal antibodies (mAbs) directed against an immunodominant region, so-called epitope cluster II, on the surface of ricins ribosome-inactivating enzymatic subunit, RTA. The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10 x LD50 ricin challenge by injection. Only three of the mAbs (LE4, PH12 and TB12) afforded protection over the seven-day study period. Neither binding affinity nor in vitro toxin-neutralizing activity could fully account for LE4, PH12 and TB12s potent in vivo activity relative to the other six mAbs. However, epitope mapping studies by hydrogen exchange-mass spectrometry (HX-MS) revealed that LE4, PH12 and TB12 shared common contact points (i.e., \"strong\" protection by HX-MS) on RTA that encompassed residues 154-164 and 62-69, which correspond to RTA -helices D-E and {beta}-strands d-e, respectively, located on the back side of RTA relative to the active site. The other six mAbs recognized overlapping epitopes on RTA but none shared the same HX-MS profile as LE4, PH12 and TB12. A high-density competition ELISA with a panel of ricin-specific single domain camelid antibodies (VHHs) indicated that even though LE4, PH12 and TB12 make contact with similar secondary motifs, they ultimately approach RTA different from angles. These results underscore how subtle differences in epitope specificity have significant impacts on the antibody functionality in vivo and have important implications in the design of immune-based countermeasures against ricin.

immunology↗