Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III–binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.

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The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904280116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13563-13572 ◽

Cited By ~ 9

Author(s):

William E. Sause ◽

Divya Balasubramanian ◽

Irnov Irnov ◽

Richard Copin ◽

Mitchell J. Sullivan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Ex Vivo ◽

Purine Biosynthesis ◽

In Vivo Studies ◽

Human Neutrophils ◽

Infection Models ◽

Genes Encoding ◽

Virulence Regulator

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR’s canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR. Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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A human antibody selective for transthyretin amyloid removes cardiac amyloid through phagocytic immune cells

Nature Communications ◽

10.1038/s41467-021-23274-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Aubin Michalon ◽

Andreas Hagenbuch ◽

Christian Huy ◽

Evita Varela ◽

Benoit Combaluzier ◽

...

Keyword(s):

Immune Cells ◽

Ex Vivo ◽

Phase 1 ◽

Hereditary Disease ◽

Elderly Subjects ◽

Human Antibody ◽

Cardiac Amyloid ◽

Healthy Elderly ◽

Ongoing Phase

AbstractTransthyretin amyloid (ATTR) cardiomyopathy is a debilitating disease leading to heart failure and death. It is characterized by the deposition of extracellular ATTR fibrils in the myocardium. Reducing myocardial ATTR load is a therapeutic goal anticipated to translate into restored cardiac function and improved patient survival. For this purpose, we developed the selective anti-ATTR antibody NI301A, a recombinant human monoclonal immunoglobulin G1. NI301A was cloned following comprehensive analyses of memory B cell repertoires derived from healthy elderly subjects. NI301A binds selectively with high affinity to the disease-associated ATTR aggregates of either wild-type or variant ATTR related to sporadic or hereditary disease, respectively. It does not bind physiological transthyretin. NI301A removes ATTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived ATTR fibrils in a dose- and time-dependent fashion. The biological activity of ATTR removal involves antibody-mediated activation of phagocytic immune cells including macrophages. These data support the evaluation of safety and tolerability of NI301A in an ongoing phase 1 clinical trial in patients with ATTR cardiomyopathy.

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A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

Scientific Reports ◽

10.1038/s41598-021-89882-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroto Nakajima ◽

Atsushi Miyashita ◽

Hiroshi Hamamoto ◽

Kazuhisa Sekimizu

Keyword(s):

Inflammatory Cytokines ◽

Immune Cells ◽

Ex Vivo ◽

Large Bubble ◽

Suitable Model ◽

Bacterial Cells ◽

Functional Impairments ◽

Gene Expressions ◽

Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

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Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival

10.1101/478628 ◽

2018 ◽

Cited By ~ 2

Author(s):

Nicholas Borcherding ◽

Kawther K. Ahmed ◽

Andrew P. Voigt ◽

Ajaykumar Vishwakarma ◽

Ryan Kolb ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Immune Cells ◽

Ex Vivo ◽

Expression Patterns ◽

Suppressive Effect ◽

Renal Clear Cell Carcinoma ◽

Cell Fates ◽

Treg Subset

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

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Rabbit model of Staphylococcus aureus implant-associated spinal infection

Disease Models & Mechanisms ◽

10.1242/dmm.045385 ◽

2020 ◽

Vol 13 (7) ◽

pp. dmm045385

Author(s):

Oren Gordon ◽

Robert J. Miller ◽

John M. Thompson ◽

Alvaro A. Ordonez ◽

Mariah H. Klunk ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bone Remodeling ◽

Ex Vivo ◽

Rabbit Model ◽

Pedicle Screws ◽

Lumbar Vertebra ◽

Wound Dehiscence ◽

Spinal Infection ◽

Positron Emission

ABSTRACTPost-surgical implant-associated spinal infection is a devastating complication commonly caused by Staphylococcus aureus. Biofilm formation is thought to reduce penetration of antibiotics and immune cells, contributing to chronic and difficult-to-treat infections. A rabbit model of a posterior-approach spinal surgery was created, in which bilateral titanium pedicle screws were interconnected by a plate at the level of lumbar vertebra L6 and inoculated with a methicillin-resistant S.aureus (MRSA) bioluminescent strain. In vivo whole-animal bioluminescence imaging (BLI) and ex vivo bacterial cultures demonstrated a peak in bacterial burden by day 14, when wound dehiscence occurred. Structures suggestive of biofilm, visualized by scanning electron microscopy, were evident up to 56 days following infection. Infection-induced inflammation and bone remodeling were also monitored using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and computed tomography (CT). PET imaging signals were noted in the soft tissue and bone surrounding the implanted materials. CT imaging demonstrated marked bone remodeling and a decrease in dense bone at the infection sites. This rabbit model of implant-associated spinal infection provides a valuable preclinical in vivo approach to investigate the pathogenesis of implant-associated spinal infections and to evaluate novel therapeutics.

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Polyketides from marine-derived Aspergillus welwitschiae inhibit Staphylococcus aureus virulence factors and potentiate vancomycin antibacterial activity in vivo

Microbial Pathogenesis ◽

10.1016/j.micpath.2020.104066 ◽

2020 ◽

Vol 143 ◽

pp. 104066 ◽

Cited By ~ 1

Author(s):

Luciana A. Loges ◽

Denise B. Silva ◽

Gustavo V.B. Paulino ◽

Melissa F. Landell ◽

Alexandre J. Macedo

Keyword(s):

Staphylococcus Aureus ◽

Antibacterial Activity ◽

Virulence Factors

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Enhancement in Site-Specific Delivery of Carvacrol against Methicillin Resistant Staphylococcus aureus Induced Skin Infections Using Enzyme Responsive Nanoparticles: A Proof of Concept Study

Pharmaceutics ◽

10.3390/pharmaceutics11110606 ◽

2019 ◽

Vol 11 (11) ◽

pp. 606 ◽

Cited By ~ 8

Author(s):

Maria Mir ◽

Naveed Ahmed ◽

Andi Dian Permana ◽

Aoife Maria Rodgers ◽

Ryan F. Donnelly ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Ex Vivo ◽

Infection Model ◽

Skin Infections ◽

Site Specific ◽

Methicillin Resistant ◽

Hydrogel Matrix ◽

Skin Retention

Methicillin resistant Staphylococcus aureus (MRSA) induced skin infections have become a challenging problem due to the escalating antibiotic resistance. Carvacrol (CAR) has been reported to be effective against MRSA. However, due to its characteristics, CAR exhibits low skin retention. In this study, CAR was formulated into site-specific nanoparticle (NPs) delivery system using poly(ε-caprolactone) (PCL), following incorporation into a hydrogel matrix to facilitate dermal delivery. The release study exhibited significantly higher release of CAR from PCL NPs in the presence of bacterial lipase, highlighting its potential for differential delivery. Moreover, encapsulation of CAR in PCL NPs resulted in a two-fold increase in its anti-MRSA activity. Dermatokinetic studies revealed that the NPs loaded hydrogel was able to enhance skin retention of CAR after 24 h (83.29 ± 3.15%), compared to free CAR-loaded hydrogel (0.85 ± 0.14%). Importantly, this novel approach exhibited effective antimicrobial activity in an ex-vivo skin infection model. Hence, these findings have proven the concept that the loading of CAR into a responsive NPs system can lead to sustained antimicrobial effect at the desired site, and may provide a novel effective approach for treatment of MRSA induced skin infections. However, further studies must be conducted to investigate in-vivo efficacy of the developed system in an appropriate infection model.

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Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Infection and Immunity ◽

10.1128/iai.00293-20 ◽

2020 ◽

Vol 88 (11) ◽

Author(s):

Marloes I. Hofstee ◽

Martijn Riool ◽

Igors Terjajevs ◽

Keith Thompson ◽

Martin J. Stoddart ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Cells ◽

Early Stage ◽

Three Dimensional ◽

Collagen Gel ◽

Maximum Size ◽

Human Neutrophils ◽

Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

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Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-Scid, IL-2Rγnull Mice In Vivo.

Blood ◽

10.1182/blood.v114.22.726.726 ◽

2009 ◽

Vol 114 (22) ◽

pp. 726-726

Author(s):

Asahi Ito ◽

Takashi Ishida ◽

Atae Utsunomiya ◽

Fumihiko Sato ◽

Fumiko Mori ◽

...

Keyword(s):

Mouse Model ◽

Tumor Cells ◽

Cell Lines ◽

Immune Cells ◽

Treg Cells ◽

Humanized Mice ◽

Effector Cells ◽

Human Immune ◽

Nog Mice

Abstract Abstract 726 There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities, and it is a current crucial problem in the field of human ADCC research. To overcome this, we have established “humanized mice,” in which human immune cells from healthy individuals function as ADCC effector cells against allogeneic tumor cell lines, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients. In this model, the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to highly enhance ADCC, showed potent antitumor activity by human ADCC against CCR4 expressing tumor cell lines. In addition, KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in the tumor bearing humanized mice. These observations indicate that KM2760 could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. Using this humanized mouse model, we now have the opportunity to perform more appropriate preclinical evaluation of many types of mAb based immunotherapy, although in the initial study, we could not completely exclude nonspecific allogeneic immune responses because target and effector cells were obtained from different individuals. In addition, susceptibility to immunotherapy is likely to be different in established cell lines and primary tumor cells isolated directly ex vivo from patients, with the latter certainly being more relevant for evaluation of immunotherapeutic agents. To overcome the subsequent problems, we have established a primary human tumor bearing NOG mouse model, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T-cell leukemia/lymphoma (ATLL) cells. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in mAb based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.

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