scholarly journals Monoclonal antibodies opsonise Burkholderia and reduce intracellular actin tail formation in a macrophage infection assay.

2021 ◽  
Author(s):  
A. Taylor ◽  
D. Jenner ◽  
C. Rowland ◽  
T. Laws ◽  
I. Norville ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Burkholderia Pseudomallei ◽  
Bacterial Virulence ◽  
Neglected Tropical Disease ◽  
Intracellular Pathogen ◽  
Murine Macrophage ◽  
Macrophage Infection ◽  
Spread Of Infection ◽  
Selection Of

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei . The bacterium is intrinsically resistant to various antibiotics and melioidosis is therefore difficult to treat successfully without relapse in infection. B. pseudomallei is an intracellular pathogen, and therefore to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies to opsonise Burkholderia species and determine the effect that the antibody has on bacterial virulence in vitro . Murine macrophage infection assays demonstrated that monoclonal antibodies to the capsule of B. pseudomallei are opsonising. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating antibodies could reduce the intracellular spread of B. thailandensis . The data presented in this paper demonstrates that monoclonal antibodies are opsonising and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. This data has informed selection of an antibody for development of an antibody-antibiotic conjugate for melioidosis. Importance statement Melioidosis is difficult to treat successfully due to the bacterium being resistant to many classes of antibiotics, therefore available therapeutic options are limited. New and improved therapies are urgently required to treat this disease. Here we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies we hope to disarm the pathogen, reducing the spread of infection. Ultimately we aim to use an opsonising antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

scholarly journals Antibiotic Tolerance: Distinguishing between Classical Resistance and Persistence in a Macrophage Infection Model

Fine Focus ◽  
2018 ◽  
Vol 4 (1) ◽  
pp. 117-129
Author(s):  
Crystal Collins ◽  
Kara Mosovsky
Keyword(s):  
Burkholderia Pseudomallei ◽  
Infection Model ◽  
Intracellular Pathogen ◽  
Macrophage Infection ◽  
Persister Cells ◽  
Antibiotic Resistant ◽  
Related Organism ◽  
Infection Models ◽  
High Doses

Burkholderia pseudomallei is a Gram-negative bacillus and facultative intracellular pathogen. It causes the disease melioidosis, which is a potentially fatal human disease found throughout the world but particularly in Southeast Asia and Northern Australia. B. pseudomallei is inherently antibiotic resistant and therefore new therapies are needed to combat this pathogen. Previous studies with the related organism Burkholderia thailandensis have shown that the antibiotic ceftazidime does not eliminate all bacteria in an in vitro macrophage model, and the remaining bacteria could still pose a health threat to a potential host. Due to their survival in the presence of antibiotics, we hypothesized that the remaining bacteria were one of two types of antibiotic tolerant cells: classically antibiotic resistant cells or persister cells. To test our hypothesis we isolated the bacteria that had survived ceftazidime treatment in the macrophage infection model and performed additional in vitro experiments to show that the surviving bacteria are neither antibiotic resistant nor persister cells. Instead, they are still susceptible to high doses (200 μg/ml) of the antibiotic over a period of 48 hours (p<0.001). We believe the bacteria survive exposure to the antibiotic during the macrophage infection because of their ability to move between intracellular and extracellular compartments, thus avoiding the antibiotic and its deadly effects. Our results provide evidence to suggest that intracellular pathogens, through movement between intracellular and extracellular compartments, may be protected from the effects of antibiotics in similar macrophage infection models.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

scholarly journals Modified Virulence of Antibiotic-Induced Burkholderia pseudomallei Filaments

2005 ◽  
Vol 49 (3) ◽  
pp. 1002-1009 ◽  
Author(s):  
Kang Chen ◽  
Guang Wen Sun ◽  
Kim Lee Chua ◽  
Yunn-Hwen Gan
Keyword(s):  
Burkholderia Pseudomallei ◽  
Bacterial Virulence ◽  
In Vitro Assays ◽  
Filamentous Bacteria ◽  
Antibiotic Concentration ◽  
Temporary Reduction ◽  
Monocytic Cells ◽  
Life Threatening ◽  
Sublethal Concentrations

ABSTRACT Melioidosis is a life-threatening bacterial infection caused by Burkholderia pseudomallei. Some antibiotics used to treat melioidosis can induce filamentation in B. pseudomallei. Despite studies on the mechanism of virulence of the bacteria, the properties of B. pseudomallei filaments and their impact on virulence have not been investigated before. To understand the characteristics of antibiotic-induced filaments, we performed in vitro assays to compare several aspects of virulence between normal, nonfilamentous and filamentous B. pseudomallei. Normal, nonfilamentous B. pseudomallei could cause the lysis of monocytic cells, while filaments induced by sublethal concentrations of ceftazidime, ofloxacin, or trimethoprim show decreased lysis of monocytic cells, especially after prolonged antibiotic exposure. The motility of the filamentous bacteria was reduced compared to that of nonfilamentous bacteria. However, the filamentation was reversible when the antibiotics were removed, and the revertant bacteria recovered their motility and ability to lyse monocytic cells. Meanwhile, antibiotic resistance developed in revertant bacteria exposed to ceftazidime at the MIC. Our study highlights the danger of letting antibiotic concentration drop to the MIC or sub-MICs during antibiotic treatment of melioidosis. This could potentially give rise to a temporary reduction of bacterial virulence, only to result in bacteria that are equally virulent but more resistant to antibiotics, should the antibiotics be reduced or removed.

scholarly journals Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e90930 ◽  
Author(s):  
Bhavani V. Peddayelachagiri ◽  
Soumya Paul ◽  
Shivakiran S. Makam ◽  
Radhika M. Urs ◽  
Joseph J. Kingston ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Burkholderia Pseudomallei ◽  
Protective Efficacy ◽  
Functional Characterization ◽  
Murine Monoclonal Antibodies

scholarly journals Manufacturing of hybridomas, producing monoclonal antibodies against Burkholderia mallei and Burkholderia pseudomallei antigens

2019 ◽  
pp. 78-82
Author(s):  
G. V. Kuklina ◽  
G. D. Elagin ◽  
D. V. Pechenkin ◽  
O. O. Fomenkov ◽  
A. V. Eremkin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Lymphocytes ◽  
Burkholderia Pseudomallei ◽  
Specific Activity ◽  
Burkholderia Mallei ◽  
Causative Agents ◽  
Biological Tests ◽  
Diagnostic Capabilities

Aim. Obtaining hybridomas, stable producing specific monoclonal antibodies against Burkholderia mallei and Burkholderia pseudomallei antigens. Materials and methods. The microbial cultures from State Collection of Microorganisms from the Branch of 48 CSRI of the Defense Ministry of Russian Federation (Kirov) and BALB/c mouse were used in research. Hybridization of B lymphocytes with SP2/0-Ag14 myeloma cells was performed by G.Kohler and C.Milstein procedure in De St. Fazekas and D.Scheidegger modification. The specific activity of immune sera, hybridoma supernatants, ascites and evaluating the diagnostic capabilities of monoclonal antibodies was studied by ELISA. Results. Hybridomas, producing monoclonal antibodies against causative agents of glanders and melioidosis antigens, were obtained and characterized. Obtained hybridomas are active and stable antibody producers after repeated in vitro and in vivo passaging. Immunoglobulins from obtained ascites were isolated. Antibodies provided the greatest sensitivity and specificity were selected. Conclusion. Monoclonal antibodies, producing by obtained hybridomas may be used for creating of immune biological tests.

scholarly journals Agglutination ofHistoplasma capsulatumby IgG Monoclonal Antibodies against Hsp60 Impacts Macrophage Effector Functions

2010 ◽  
Vol 79 (2) ◽  
pp. 918-927 ◽  
Author(s):  
Allan Jefferson Guimarães ◽  
Susana Frases ◽  
Bruno Pontes ◽  
Mariana Duarte de Cerqueira ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Optical Tweezers ◽  
Histoplasma Capsulatum ◽  
Yeast Cells ◽  
Macrophage Infection ◽  
Cell Agglutination ◽  
Effector Functions ◽  
Antigen Epitope ◽  
Antibody Subclass

ABSTRACTHistoplasma capsulatumcan efficiently survive within macrophages, facilitatingH. capsulatumtranslocation from the lung into the lymphatics and bloodstream. We have recently generated monoclonal antibodies (MAbs) to anH. capsulatumsurface-expressed heat shock protein of 60 kDa (Hsp60) that modify disease in a murine histoplasmosis model. Interestingly, the MAbs induced different degrees of yeast cell agglutinationin vitro. In the present study, we characterized the agglutination effects of the antibodies to Hsp60 onH. capsulatumyeast cells by light microscopy, flow cytometry, dynamic light scattering, measuring zeta potential, and using optical tweezers. We found that immunoglobulin Gs (IgGs) to Hsp60 causeH. capsulatumaggregation dependent on the (i) concentration of MAbs, (ii) MAb binding constant, and (iii) IgG subclass. Furthermore, infection of macrophages using agglutinates of various sizes after incubation with different Hsp60-binding MAbs induced association to macrophages through distinct cellular receptors and differentially affected macrophage antifungal functions. Hence, the capacity of IgG MAbs to agglutinateH. capsulatumsignificantly impacted pathogenic mechanisms ofH. capsulatumduring macrophage infection, and the effect was dependent on the antibody subclass and antigen epitope.

scholarly journals Evaluation of the Antibacterial Activity of Piperacillin–tazobactam Against Burkholderia Pseudomallei in Vitro

2021 ◽  
Author(s):  
Mei hui Huang ◽  
Hua Wu ◽  
Xiao jun Zhou ◽  
Qin fang Cao ◽  
Xu ming Wang
Keyword(s):  
Antibacterial Activity ◽  
Clavulanic Acid ◽  
Theoretical Basis ◽  
Burkholderia Pseudomallei ◽  
Therapeutic Options ◽  
Antimicrobial Sensitivity ◽  
Amoxicillin Clavulanic Acid ◽  
First And Second Generation ◽  
Selection Of

Abstract Objective:Melioidosis is a zoonotic disease caused by Burkholderia pseudomallei (B. pseudomallei). Because B. pseudomallei is naturally resistant to a variety of antibiotics, such as penicillin, ampicillin, first-and second-generation cephalosporins, macrolides, aminoglycosides, streptomycin, polymyxin, etc., the selection of drugs is limited. The present study aimed to evaluate the antibacterial activity of piperacillin/tazobactam against B. pseudomallei in vitro to provide a theoretical basis and a variety of therapeutic options for clinical application.The antimicrobial susceptibilities of all B. pseudomallei isolated from melioidosis patients were detected by Etest method. Results: The MIC50 values of piperacillin/tazobactam, imipenem, meropenem, doxycycline, trimethoprim–sulfamethoxazole, ceftazidime, tetracycline, and amoxicillin/clavulanic acid were 0.25 μg/ml, 0.5 μg/ml, 0.5 μg/ml, 0.5 μg/ml, 1 μg/ml, 1 μg/ml, 2 μg/ml, and 2 μg/ml, respectively. Their MIC90 values were 0.5 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 2 μg/ml, 2 μg/ml, 4 μg/ml, and 4 μg/ml, respectively. All the isolates were uniformly sensitive (100%) to ceftazidime, imipenem and amoxicillin–clavulanic acid. The rate of antimicrobial sensitivity for doxycycline was 99.08%, for tetracycline-sulfamethoxazole 94.50%, and for tetracycline 94.49%.Both the MIC50 and MIC90 values of piperacillin/tazobactam were lower than those of the seven other antibiotics, indicating that it has strong antibacterial activity against B. pseudomallei in vitro.

scholarly journals Major histocompatibility complex antigen expression on the epithelium of the developing thymus in normal and nude mice.

1981 ◽  
Vol 153 (2) ◽  
pp. 280-292 ◽  
Author(s):  
E J Jenkinson ◽  
W Van Ewijk ◽  
J J Owen
Keyword(s):  
Monoclonal Antibodies ◽  
Major Histocompatibility Complex ◽  
Nude Mice ◽  
Antigen Expression ◽  
Major Histocompatibility Complex Antigen ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Selection Of

The expression and distribution of antigens coded by the K and I regions of the major histocompatibility complex in the developing thymus of normal and nude mice has been investigated using monoclonal antibodies. Both immunohistological studies of intact rudiments and in vitro labeling of cultures derived from microdissected rudiments indicate that, while K region antigens are present on epithelial and mesenchymal elements, I region antigens are only detectable on the epithelium. This view is also substantiated by the selective absence of I region antigens in the abnormal nude thymic rudiment where the defect is considered to be epithelial in nature. The findings are considered in relation to the role of the thymus in providing an environment for the differentiation and selection of developing T cells, and it is proposed that the Ia-expressing epithelial elements play a central role in these functions.

scholarly journals Cryptococcus neoformans Is a Facultative Intracellular Pathogen in Murine Pulmonary Infection

2000 ◽  
Vol 68 (7) ◽  
pp. 4225-4237 ◽  
Author(s):  
Marta Feldmesser ◽  
Yvonne Kress ◽  
Phyllis Novikoff ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Pulmonary Infection ◽  
Pathogenic Fungus ◽  
Microbial Pathogens ◽  
Yeast Cells ◽  
Intracellular Pathogen ◽  
Phagocytic Cells ◽  
Macrophage Infection

ABSTRACT To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungusCryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformansoccupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.

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