scholarly journals Binding of the von Willebrand Factor A Domain of Capillary Morphogenesis Protein 2 to Anthrax Protective Antigen Vaccine Reduces Immunogenicity in Mice

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Fabiana Freire Mendes de Oliveira ◽  
Sireesha Mamillapalli ◽  
Srinivas Gonti ◽  
Robert N. Brey ◽  
Han Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Von Willebrand Factor ◽  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Antibody Responses ◽  
Anthrax Toxin ◽  
Anthrax Vaccine ◽  
Capillary Morphogenesis ◽  
Von Willebrand ◽  
Willebrand Factor

ABSTRACT Protective antigen (PA) is a component of anthrax toxin that can elicit toxin-neutralizing antibody responses. PA is also the major antigen in the current vaccine to prevent anthrax, but stability problems with recombinant proteins have complicated the development of new vaccines containing recombinant PA. The relationship between antigen physical stability and immunogenicity is poorly understood, but there are theoretical reasons to think that this parameter can affect immune responses. We investigated the immunogenicity of anthrax PA, in the presence and absence of the soluble von Willebrand factor A domain of the human form of receptor capillary morphogenesis protein 2 (sCMG2), to elicit antibodies to PA in BALB/c mice. Prior studies showed that sCMG2 stabilizes the 83-kDa PA structure to pH, chemical denaturants, temperature, and proteolysis and slows the hydrogen-deuterium exchange rate of histidine residues far from the binding interface. In contrast to a vaccine containing PA without adjuvant, we found that mice immunized with PA in stable complex with sCMG2 showed markedly reduced antibody responses to PA, including toxin-neutralizing antibodies and antibodies to domain 4, which correlated with fewer toxin-neutralizing antibodies. In contrast, mice immunized with PA in concert with a nonbinding mutant of sCMG2 (D50A) showed anti-PA antibody responses similar to those observed with PA alone. Our results suggest that addition of sCMG2 to a PA vaccine formulation is likely to result in a significantly diminished immune response, but we discuss the multitude of factors that could contribute to reduced immunogenicity. IMPORTANCE The anthrax toxin PA is the major immunogen in the current anthrax vaccine (anthrax vaccine adsorbed). Improving the anthrax vaccine for avoidance of a cold chain necessitates improvements in the thermodynamic stability of PA. We address how stabilizing PA using sCMG2 affects PA immunogenicity in BALB/c mice. Although the stability of PA is increased by binding to sCMG2, PA immunogenicity is decreased. This study emphasizes that, while binding of a ligand retains or improves conformational stability without affecting the native sequence, epitope recognition or processing may be affected, abrogating an effective immune response.

scholarly journals Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Joseph Jelinski ◽  
Austen Terwilliger ◽  
Sabrina Green ◽  
Anthony Maresso
Keyword(s):  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Anthrax Toxin ◽  
Cell Binding ◽  
Lethal Challenge ◽  
Anthrax Vaccine ◽  
Content Type ◽  
Heme Transport ◽  
Bacillus Spores ◽  
Culture Supernatants

ABSTRACT Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund’s Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Detection of Non Inhibitory Binding Antibodies to Von Willebrand Factor Affecting the Clearance of VWF:Ag in Von Willebrand Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2275-2275
Author(s):  
Tobias M Suiter ◽  
Pier Mannuccio Mannucci ◽  
Christine L Kempton ◽  
Michael Laffan ◽  
Edward H Romond ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Von Willebrand Disease ◽  
Inhibitory Antibody ◽  
Screening Assay ◽  
Post Infusion ◽  
Inhibitory Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2275 Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF, the largest multimeric plasma glycoprotein, facilitates platelet aggregation and stabilizes FVIII in the circulation. Inhibitory antibodies to VWF have been reported in 10% – 15% of type 3 VWD patients repetitively treated with plasma-derived VWF/FVIII concentrates. The clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), both total binding anti-VWF antibodies and inhibitory anti-VWF antibodies (VWF:Ristocetin Cofactor Activity [RCo], VWF:Collagen Binding Activity [CB], VWF:FVIII Binding [FVIIIB]) were assessed in a Phase 1 multicenter clinical study. Total binding anti-VWF antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) employing polyclonal anti-human IgG antibodies. Plasma samples were analyzed in two steps employing a screening assay determining the titer of the binding antibodies and in a second step, confirming the specificity of the antibody in a competition assay. Samples were considered positive, when a sample was at least two titer steps lower than the antibody titer detected in the screening assay necessitating a screening titer of at least 1:80. Neutralizing antibodies to the key functional activities of VWF, such as VWF:RCo, VWF:CB and VWF:FVIIIB, were measured by assays based on the Bethesda assay established for quantitative analysis of FVIII inhibitors (Nijmegen modification of the Bethesda assay). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all 3 assays. Three of 39 subjects screened had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF. One of these 3 subjects was excluded from the study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening. Two of the 3 subjects were enrolled and treated with rVWF and/or pdVWF concentrate as part of the safety, tolerability and PK assessments. The high titer binding anti-VWF antibodies were associated with a significant decreased VWF:Ag activity post infusion of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. For example, one of the subject had a reduced VWF:Ag incremental recovery of 1.1 IU/dL/(U VWF:RCo/kg) (mean of cohort 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t1/2 2.4 hours (mean of cohort t1/2 25.3 hours) post infusion of rhVWF dosed at 50 IU VWF:RCo/kg. Relevant data of all PK assessments and antibody titers will be presented. None of the study subjects developed an inhibitory antibody to either VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates noted by the treating physician. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation. Disclosures: Suiter: Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment. Varadi:Baxter Innovations GmbH: Employment. Chapman:Baxter Innovations GmbH: Employment. Engl:Baxter Innovations GmbH: Employment. Wong:Baxter Healthcare Corp: Employment. Ewenstein:Baxter Healthcare Corp: Employment.

scholarly journals Effect of Anthrax Immune Globulin on Response to BioThrax (Anthrax Vaccine Adsorbed) in New Zealand White Rabbits

2013 ◽  
Vol 57 (11) ◽  
pp. 5693-5696 ◽  
Author(s):  
Nina V. Malkevich ◽  
Subhendu Basu ◽  
Thomas L. Rudge ◽  
Kristin H. Clement ◽  
Ajoy C. Chakrabarti ◽  
...  
Keyword(s):  
Immune Response ◽  
New Zealand ◽  
Immune Globulin ◽  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Anthrax Vaccine ◽  
New Zealand White Rabbits ◽  
Anthrax Toxins

ABSTRACTDevelopment of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.

Von Willebrand Factor Modulates Factor VIII Immunogenicity: Comparative Study of Different Factor VIII Concentrates in a Haemophilia A Mouse Model

2002 ◽  
Vol 88 (08) ◽  
pp. 221-229 ◽  
Author(s):  
Mathias Behrmann ◽  
John Pasi ◽  
Jean-Marie Saint-Remy ◽  
Ronald Kotitschke ◽  
Michael Kloft
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Major Complication ◽  
Epidemiological Studies ◽  
Haemophilia A ◽  
Epitope Specificity ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe development of an immune response towards factor VIII (FVIII) remains the major complication of haemophilia A replacement therapy. Product-related risk factors have recently been identified on the basis of epidemiological studies, but the mechanism is not understood. To this end, various commercially available FVIII concentrates were administered by the IV route to FVIII-knockout mice and the resulting immune response was characterised. Significantly higher inhibitor titres (Bethesda assay) were observed for one recombinant FVIII and one plasma-derived FVIII product depleted in von Willebrand factor (VWF). Inhibitor titres were reduced upon pre-incubation of FVIII with purified VWF. Epitope specificity of anti-FVIII IgG was characterised using FVIII-fragments produced in E. coli. Concentrates with no or reduced VWF-level elicited antibodies recognising predominantly the acidic a1 and a3 regions. Addition of VWF prior to injection also modified the epitope specificity. FVIII concentrates, therefore, show qualitative and quantitative variations in immunogenicity, which are at least partly modulated by VWF.

von Willebrand factor: what is its role in the immune response in haemophilia?

Haemophilia ◽  
2010 ◽  
Vol 17 (1) ◽  
pp. e235-e238 ◽  
Author(s):  
S. KAVERI ◽  
P. M. MANNUCCI ◽  
M. H. KURTH ◽  
N. EWING ◽  
C. M. KESSLER ◽  
...  
Keyword(s):  
Immune Response ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Neutralizing Activity of Vaccine-Induced Antibodies to Two Bacillus anthracis Toxin Components, Lethal Factor and Edema Factor

2007 ◽  
Vol 15 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Sarah C. Taft ◽  
Alison A. Weiss
Keyword(s):  
Animal Models ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Antibody Responses ◽  
Anthrax Toxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mouse Macrophage ◽  
Anthrax Vaccine ◽  
Neutralizing Activity ◽  
Edema Factor

ABSTRACT Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.

scholarly journals A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake

2019 ◽  
Author(s):  
Jordan G. Finnell ◽  
Tsz-Ming Tsang ◽  
Lorna Cryan ◽  
Samuel Garrard ◽  
Sai-Lun Lee ◽  
...  
Keyword(s):  
Metal Ion ◽  
Protective Antigen ◽  
Anthrax Toxin ◽  
Peptide Array ◽  
Peptide Fragments ◽  
Capillary Morphogenesis ◽  
Type Iv ◽  
Toxin Receptor ◽  
Affinity Peptide ◽  
Von Willebrand

AbstractCapillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. In addition to its role as an anthrax toxin receptor, CMG2 has been repeatedly shown to play a role in angiogenic processes. However, the molecular mechanism mediating observed CMG2-related angiogenic effects has not been fully elucidated. Previous studies have found that CMG2 binds type IV collagen (Col-IV), a key component of the vascular basement membrane, as well as other ECM proteins. Currently, no link has been made between these CMG2-ECM interactions and angiogenesis; however, ECM fragments are known to play a role in regulating angiogenesis. Here, we further characterize the CMG2-Col-IV interaction and explore the effect of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (non-collagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative binding epitope. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with sub-micromolar affinity (peptide S16, Kd = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding, and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. We demonstrate that CMG2 specifically mediates endocytic uptake of S16, since CMG2-/- endothelial cells show markedly reduced S16 uptake, without reducing total endocytosis. Furthermore, we show that S16 reduces endothelial migration but not cell proliferation. Taken together, our data demonstrate that a Col IV-derived anti-angiogenic peptide acts via CMG2, suggesting a possible link between CMG2-Col IV interactions and angiogenesis.

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