scholarly journals Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

2015 ◽  
Vol 22 (5) ◽  
pp. 553-560 ◽  
Author(s):  
Xiangyang Chi ◽  
Jianmin Li ◽  
Weicen Liu ◽  
Xiaolin Wang ◽  
Kexin Yin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Synergistic Effects ◽  
Central Component ◽  
Dependent Manner ◽  
Human Monoclonal Antibodies ◽  
Specific Antibodies ◽  
Anthrax Protective Antigen

ABSTRACTThe anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the “next-generation” recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection withBacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) bothin vitroandin vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective onlyin vitrowas found to be neutralizingin vivoin combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

scholarly journals High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Hong Feng ◽  
Junling Gu ◽  
Fang Gou ◽  
Wei Huang ◽  
Chenlin Gao ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Nlrp3 Inflammasome ◽  
High Glucose ◽  
Mesangial Cells ◽  
Central Component ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Interacting Protein

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1βwas observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1βwere significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1βinflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Antitumor effects of the small molecule DMAMCL in neuroblastoma via suppressing aerobic glycolysis and targeting PFKL

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Simeng Zhang ◽  
Zhongyan Hua ◽  
Gen Ba ◽  
Ning Xu ◽  
Jianing Miao ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effects ◽  
Aerobic Glycolysis ◽  
Single Agent ◽  
Molecular Compound ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Atp Production

Abstract Background Neuroblastoma (NB) is a common solid malignancy in children that is associated with a poor prognosis. Although the novel small molecular compound Dimethylaminomicheliolide (DMAMCL) has been shown to induce cell death in some tumors, little is known about its role in NB. Methods We examined the effect of DMAMCL on four NB cell lines (NPG, AS, KCNR, BE2). Cellular confluence, survival, apoptosis, and glycolysis were detected using Incucyte ZOOM, CCK-8 assays, Annexin V-PE/7-AAD flow cytometry, and Seahorse XFe96, respectively. Synergistic effects between agents were evaluated using CompuSyn and the effect of DMAMCL in vivo was evaluated using a xenograft mouse model. Phosphofructokinase-1, liver type (PFKL) expression was up- and down-regulated using overexpression plasmids or siRNA. Results When administered as a single agent, DMAMCL decreased cell proliferation in a time- and dose-dependent manner, increased the percentage of cells in SubG1 phase, and induced apoptosis in vitro, as well as inhibiting tumor growth and prolonging survival in tumor-bearing mice (NGP, BE2) in vivo. In addition, DMAMCL exerted synergistic effects when combined with etoposide or cisplatin in vitro and displayed increased antitumor effects when combined with etoposide in vivo compared to either agent alone. Mechanistically, DMAMCL suppressed aerobic glycolysis by decreasing glucose consumption, lactate excretion, and ATP production, as well as reducing the expression of PFKL, a key glycolysis enzyme, in vitro and in vivo. Furthermore, PFKL overexpression attenuated DMAMCL-induced cell death, whereas PFKL silencing promoted NB cell death. Conclusions The results of this study suggest that DMAMCL exerts antitumor effects on NB both in vitro and in vivo by suppressing aerobic glycolysis and that PFKL could be a potential target of DMAMCL in NB.

scholarly journals Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Xuyao Jiao ◽  
Sarah Smith ◽  
Gabrielle Stack ◽  
Qi Liang ◽  
Allan Bradley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Typhoid Fever ◽  
Severe Disease ◽  
Genetically Engineered ◽  
Human Monoclonal Antibodies ◽  
Genetically Engineered Mice ◽  
Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

scholarly journals Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies

2004 ◽  
Vol 72 (11) ◽  
pp. 6313-6317 ◽  
Author(s):  
Fabien Brossier ◽  
Martine Lévy ◽  
Annie Landier ◽  
Pierre Lafaye ◽  
Michèle Mock
Keyword(s):  
Functional Analysis ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Cell Receptor ◽  
Lethal Challenge ◽  
Edema Factor

ABSTRACT Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

scholarly journals Mouse Monoclonal Antibodies to Anthrax Edema Factor Protect against Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4609-4616 ◽  
Author(s):  
Clinton E. Leysath ◽  
Kuang-Hua Chen ◽  
Mahtab Moayeri ◽  
Devorah Crown ◽  
Rasem Fattah ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Essential Element ◽  
Content Type ◽  
Time To Death ◽  
Edema Factor ◽  
Edema Toxin

ABSTRACTBacillus anthracisis the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic CBdomain. MAb 7F10 shows potent neutralization of edema toxin activityin vitroandin vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.

scholarly journals Humans surviving cholera develop antibodies against Vibrio cholerae O-specific polysaccharide that inhibit pathogen motility

2020 ◽  
Author(s):  
Richelle C. Charles ◽  
Meagan Kelly ◽  
Jenny M. Tam ◽  
Aklima Akter ◽  
Motaher Hossain ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vibrio Cholerae ◽  
Polyclonal Antibody ◽  
High Speed ◽  
Polyclonal Antibodies ◽  
Intestinal Lumen ◽  
Dependent Manner ◽  
Specific Antibodies ◽  
Specific Polysaccharide

ABSTRACTThe mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae correlate highly with protection against cholera. V. cholerae is highly motile and possesses a flagellum sheathed in O-specific polysaccharide (OSP), and motility of V. cholerae correlates with virulence. Using high speed video microscopy, and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block V. cholerae motility at both subagglutinating and agglutinating concentrations. This anti-motility effect is reversed by pre-adsorbing sera and polyclonal antibody fractions with purified OSP; and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. F[ab] fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated crosslinking in motility inhibition. We show that OSP-specific antibodies do not directly affect V. cholerae viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used in vivo competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of V. cholerae in intestinal tissues, and that this impact is motility-dependent. Our findings suggest that the impedance of motility by antibodies targeting V. cholerae OSP contributes to protection against cholera.IMPORTANCECholera is a severe dehydrating illness of humans caused by Vibrio cholerae. V. cholerae is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and V. cholerae motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since V. cholerae is a non-invasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit V. cholerae motility and are associated with protection against challenge in a motility-dependent manner.

scholarly journals Anthrax Protective Antigen Cleavage and Clearance from the Blood of Mice and Rats

2007 ◽  
Vol 75 (11) ◽  
pp. 5175-5184 ◽  
Author(s):  
Mahtab Moayeri ◽  
Jason F. Wiggins ◽  
Stephen H. Leppla
Keyword(s):  
Receptor Binding ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Dominant Negative ◽  
Furin Cleavage ◽  
Defective Mutant ◽  
Tissue Surfaces ◽  
Anthrax Protective Antigen

ABSTRACT Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.

scholarly journals Human Monoclonal Antibodies Directed against Toxins A and B Prevent Clostridium difficile-Induced Mortality in Hamsters

2006 ◽  
Vol 74 (11) ◽  
pp. 6339-6347 ◽  
Author(s):  
Gregory J. Babcock ◽  
Teresa J. Broering ◽  
Hector J. Hernandez ◽  
Robert B. Mandell ◽  
Katherine Donahue ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Human Monoclonal Antibodies ◽  
Toxin A ◽  
Hamster Model ◽  
Cytotoxic Effects ◽  
Toxin B ◽  
Reduction Of Mortality

ABSTRACT Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.

scholarly journals Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Elizabeth Diago-Navarro ◽  
Isabel Calatayud-Baselga ◽  
Donglei Sun ◽  
Camille Khairallah ◽  
Inderjit Mann ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Intravital Microscopy ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Anthrax Protective Antigen

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

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