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Biochemical Pharmacology Aug 2016Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization,... (Review)
Review
Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization, positioning and homeostatic trafficking of leukocytes; as well as their recruitment to, and their retention at, inflammatory sites. Ligand induced changes in the molecular conformation of chemoattractant receptors results in the engagement of heterotrimeric G-proteins, which promotes α subunits to undergo GTP/GDP exchange. This results in the functional release of βγ subunits from the heterotrimers, thereby activating downstream effector molecules, which initiate leukocyte polarization, gradient sensing, and directional migration. Pertussis toxin ADP ribosylates Gαi subunits and prevents chemoattractant receptors from triggering Gαi nucleotide exchange. The use of pertussis toxin revealed the essential importance of Gαi subunit nucleotide exchange for chemoattractant receptor signaling. More recent studies have identified a range of regulatory mechanisms that target these receptors and their associated heterotrimeric G-proteins, thereby helping to control the magnitude, kinetics, and duration of signaling. A failure in these regulatory pathways can lead to impaired receptor signaling and immunopathology. The analysis of mice with targeted deletions of Gαi isoforms as well as some of these G-protein regulatory proteins is providing insights into their roles in chemoattractant receptor signaling.
Topics: Animals; Chemotaxis, Leukocyte; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Immune System; Pertussis Toxin; RGS Proteins; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 27071343
DOI: 10.1016/j.bcp.2016.04.005 -
Molecular Microbiology May 2022Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind...
Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen-presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy-hydroxide (AlOOH), in RAW 264.7 murine and U-937 human macrophages-like cells. Furthermore, we determined the impact that the adsorption to AlOOH particulates has on the trafficking of a Bordetella pertussis vaccine candidate, the genetically detoxified pertussis toxin (gdPT). Our results indicate that macrophages internalize AlOOH by constitutive macropinocytosis assisted by the filopodial protrusions that capture the adjuvant particles. Moreover, we show that AlOOH has the capacity to nonspecifically adsorb IgG, engaging opsonic phagocytosis, which is a feature that may allow for more effective capture and uptake of adjuvant particles by antigen-presenting cells (APCs) at the site of vaccine administration. We found that AlOOH traffics to endolysosomal compartments that hold degradative properties. Importantly, while we show that gdPT escapes degradative endolysosomes and traffics toward the retrograde pathway, as reported for the wild-type pertussis toxin, the adsorption to AlOOH diverts gdPT to traffic to the adjuvant's lysosome-type compartments, which may be key for MHC-II-driven antigen presentation and activation of CD4 T cell. Thus, our findings establish a direct link between antigen adsorption to AlOOH and the intracellular trafficking of antigens within antigen-presenting cells and bring to light a new potential mechanism for aluminum adjuvancy. Moreover, the in-vitro single-cell approach described herein provides a general framework and tools for understanding critical attributes of other vaccine formulations.
Topics: Adjuvants, Immunologic; Aluminum; Aluminum Hydroxide; Animals; Humans; Lysosomes; Macrophages; Mice; Pertussis Toxin; Pertussis Vaccine
PubMed: 35344242
DOI: 10.1111/mmi.14900 -
Science Translational Medicine Dec 2015Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease,...
Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.
Topics: Animals; Antibodies, Monoclonal, Humanized; Bordetella pertussis; CHO Cells; Cricetulus; Disease Progression; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Immunoglobulin Variable Region; Infant; Mice; Mice, Inbred BALB C; Neutralization Tests; Papio; Pertussis Toxin; Prognosis; Vaccination; Whooping Cough
PubMed: 26631634
DOI: 10.1126/scitranslmed.aad0966 -
Toxins Feb 2023As a tribute to Louis Pasteur on the occasion of the 200th anniversary of his birth, this article summarizes the main contributions of scientists from Pasteur Institutes... (Review)
Review
As a tribute to Louis Pasteur on the occasion of the 200th anniversary of his birth, this article summarizes the main contributions of scientists from Pasteur Institutes to the current knowledge of toxins produced by . The article therefore focuses on publications authored by researchers from Pasteur Institutes and is not intended as a systematic review of toxins. Besides identifying as the causative agent of whooping cough, Pasteurians have made several major contributions with respect to the structure-function relationship of the lipo-oligosaccharide, adenylyl cyclase toxin and pertussis toxin. In addition to contributing to the understanding of these toxins' mechanisms at the molecular and cellular levels and their role in pathogenesis, scientists at Pasteur Institutes have also exploited potential applications of the gathered knowledge of these toxins. These applications range from the development of novel tools to study protein-protein interactions over the design of novel antigen delivery tools, such as prophylactic or therapeutic vaccine candidates against cancer and viral infection, to the development of a live attenuated nasal pertussis vaccine. This scientific journey from basic science to applications in the field of human health matches perfectly with the overall scientific objectives outlined by Louis Pasteur himself.
Topics: Humans; Bordetella pertussis; Whooping Cough; Pertussis Toxin; Virulence Factors, Bordetella; Adenylate Cyclase Toxin; Pertussis Vaccine
PubMed: 36977067
DOI: 10.3390/toxins15030176 -
Pathogens and Disease Aug 2016The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit...
The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.
Topics: Adenosine Triphosphate; Animals; Disease Models, Animal; Enzyme Activation; Intracellular Space; Mice; Pertussis Toxin; Protein Binding; Protein Subunits; Protein Transport
PubMed: 27369899
DOI: 10.1093/femspd/ftw065 -
Frontiers in Immunology 2023Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally...
Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin.
BACKGROUND
Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT).
METHODS
The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT.
RESULTS
We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed.
CONCLUSION
The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.
Topics: United States; Humans; Pertussis Toxin; Tetanus; Diphtheria; Hemagglutinins; Reproducibility of Results; Antibodies, Bacterial; Immunoglobulin G; Diphtheria-Tetanus-acellular Pertussis Vaccines
PubMed: 37342321
DOI: 10.3389/fimmu.2023.1190404 -
Clinical Microbiology and Infection :... Jun 2021Protection induced by acellular vaccines can be short, requiring novel immunization strategies. Objectives of this study were to evaluate safety and capacity of a... (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVES
Protection induced by acellular vaccines can be short, requiring novel immunization strategies. Objectives of this study were to evaluate safety and capacity of a recombinant pertussis toxin (PTgen) -coated Viaskin® epicutaneous patch to recall memory responses in healthy adults.
METHODS
This double-blind, placebo-controlled randomized trial (Phase I) assessed the safety and immunogenicity of PTgen administered on days 0 and 14 to healthy adults using Viaskin® patches applied directly or after epidermal laser-based skin preparation. Patch administration was followed by Boostrix®dTpa on day 42. Antibodies were assessed at days 0, 14, 28, 42 and 70.
RESULTS
Among 102 volunteers enrolled, 80 received Viaskin-PT (Viaskin-PT 25 μg (n = 25), Viaskin-PT 50 μg (n = 25), laser + Viaskin-PT 25 μg (n = 5), laser + Viaskin-PT 50 μg (n = 25)), Viaskin-placebo (n = 10) or laser + Viaskin-placebo (n = 2). Incidence of adverse events was similar across groups (any local event: 21/25 (84.0%), 24/25 (96.0%), 4/5 (80.0%), 24/25 (96.0%), 8/10 (80.0%), 10/12 (83.0%), respectively). Direct application induced no detectable response. On day 42, PT-IgG geometric mean concentrations were significantly higher following laser + Viaskin-PT 25 μg and 50 μg (139.87 (95% CI 87.30-224.10) and 121.76 (95% CI 95.04-156.00), respectively), than laser + Viaskin-placebo (59.49, 95% CI 39.37-89.90). Seroresponse rates were higher following laser + Viaskin-PT 25 μg (4/5 (80.0%), 95% CI 28.4-99.5) and 50 μg (22/25 (88.0%), 95% CI 68.8-97.5) than laser + Viaskin-placebo (0/12 (0.0%), 95% CI 0.0-26.5).
CONCLUSIONS
Viaskin-PT applied after laser-based epidermal skin preparation showed encouraging safety and immunogenicity results: anti-PT booster responses were not inferior to those elicited by Boostrix®dTpa. This study is registered at ClinicalTrials.gov (NCT03035370) and was funded by DBV Technologies.
Topics: Administration, Cutaneous; Adolescent; Adult; Antibodies, Bacterial; Double-Blind Method; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Male; Pertussis Toxin; Young Adult
PubMed: 32896653
DOI: 10.1016/j.cmi.2020.08.033 -
Frontiers in Cellular and Infection... 2022Chronic otitis media (COM) is the long-term infection and inflammation of the middle ears typically caused by upper respiratory tract pathogens that are able to ascend...
Chronic otitis media (COM) is the long-term infection and inflammation of the middle ears typically caused by upper respiratory tract pathogens that are able to ascend the Eustachian tube. Our understanding of contributing factors is limited because human otopathogens cannot naturally colonize or persist in the middle ears of mice. We recently described a natural COM in mice caused by and proposed this as an experimental system to study bacterial mechanisms of immune evasion that allow persistent infection of the middle ear. Here we describe a novel pertussis toxin (PTx)-like factor unique to , apparently acquired horizontally, that is associated with its particularly efficient persistence and pathogenesis. The catalytic subunit of this toxin, PsxA, has conserved catalytic sites and substantial predicted structural homology to pertussis toxin catalytic subunit PtxA. Deletion of the gene predicted to encode the catalytic subunit, , resulted in a significant decrease in persistence in the middle ears. The defect was not observed in mice lacking T cells, indicating that PsxA is necessary for persistence only when T cells are present. These results demonstrate the role of a novel putative toxin in the persistence of and its generation of COM. This PsxA-mediated immune evasion strategy may similarly be utilized by human otopathogens, other PTx-like toxins or alternative mechanisms to disrupt critical T cell functions necessary to clear bacteria from the middle ear. This work demonstrates that this experimental system can allow for the detailed study of general strategies and specific mechanisms that otopathogens use to evade host immune responses to persist in the middle ear to cause COM.
Topics: Animals; Bacteria; Ear, Middle; Inflammation; Mice; Otitis Media; Pertussis Toxin
PubMed: 35360099
DOI: 10.3389/fcimb.2022.795230 -
Vaccine Jul 2023Despite a decrease in infections caused by Bordetella pertussis due to COVID-19 pandemic, booster vaccination of pregnant women is still recommended to protect newborns.... (Randomized Controlled Trial)
Randomized Controlled Trial
INTRODUCTION
Despite a decrease in infections caused by Bordetella pertussis due to COVID-19 pandemic, booster vaccination of pregnant women is still recommended to protect newborns. Highly immunogenic vaccines containing genetically inactivated pertussis toxin (PT) and filamentous hemagglutinin (FHA) may generate comparable anti-PT antibody concentrations, even at lower doses, to chemically inactivated acellular pertussis vaccines (Tdap) shown effective for maternal immunization.
METHODS
This phase 2 randomized, observer-blind, active-controlled non-inferiority trial was conducted in healthy Thai pregnant women randomly assigned to receive one dose of low-dose recombinant pertussis-only vaccine containing 1 µg PT and 1 µg FHA (ap1), or tetanus, reduced-dose diphtheria combined with ap1 (Tdap1), or combined with 2 µg PT and 5 µg FHA (Tdap2), or with 5 µg PT and 5 µg FHA (TdaP5, Boostagen®) or comparator containing 8 µg of chemically inactivated pertussis toxoid, 8 µg FHA, and 2.5 µg pertactin (Boostrix™, Tdap8). Blood was collected at Day 0 and Day 28 post-vaccination. The non-inferiority of the study vaccines was assessed based on anti-PT IgG antibody levels on Day 28 pooled with results from a similarly structured previous trial in non-pregnant women.
RESULTS
400 healthy pregnant women received one dose of vaccine. Combined with data from 250 non-pregnant women, all study vaccines containing PT were non-inferior to comparator vaccine (Tdap8). Both ap1 and TdaP5 vaccines could be considered to have superior immunogenicity to Tdap8. Local and systemic solicited reactions were similar among all vaccine groups.
CONCLUSIONS
Vaccine formulations containing PT were safe and immunogenic in pregnant women. The ap1 vaccine, with the lowest cost and reactogenicity, may be suitable for use in pregnant women when diphtheria and tetanus toxoids are not needed. This study is registered in the Thai Clinical Trial Registry (www.
CLINICALTRIALS
in.th), number TCTR20180725004.
Topics: Infant, Newborn; Humans; Female; Pertussis Toxin; Whooping Cough; Tetanus; Diphtheria; Pandemics; COVID-19; Pertussis Vaccine; Immunization, Secondary; Tetanus Toxoid; Vaccines, Synthetic; Antibodies, Bacterial; Diphtheria-Tetanus-acellular Pertussis Vaccines; Diphtheria-Tetanus-Pertussis Vaccine
PubMed: 37330371
DOI: 10.1016/j.vaccine.2023.06.001 -
Journal of Neuroinflammation Feb 2023Previous reports have indicated that disrupting the Wnt/β-catenin pathway in dendritic cells (DCs) may affect the progression of autoimmune inflammation; however, the...
BACKGROUND
Previous reports have indicated that disrupting the Wnt/β-catenin pathway in dendritic cells (DCs) may affect the progression of autoimmune inflammation; however, the factors and timing that regulate Wnt/β-catenin signaling have not been clearly understood.
METHODS
Experimental autoimmune uveitis (EAU) mice and Vogt-Koyanagi-Harada disease (VKH) patient samples were used to detect the expression of Wnt/β-catenin pathway genes. Western blot, real-time PCR, flow cytometry, and ELISA were performed to examine the expression of components of the Wnt/β-catenin pathway and inflammatory factors. DC-specific β-catenin knockout mice and 6-bromoindirubin-3'-oxime (BIO) administered mice were used to observe the effect of disrupting the Wnt pathway on EAU pathogenesis.
RESULTS
Wnt/β-catenin signaling was inhibited in DCs during the induction phase of EAU. The inhibition was mediated by pertussis toxin (PTX), which promoted DC maturation, in turn promoting pathogenic T cell proliferation and differentiation. In vivo experiments confirmed that deleting β-catenin in DCs enhanced EAU severity, and pre-injection of PTX advanced EAU onset. Administration of a Wnt activator (BIO) limited the effects of PTX, in turn ameliorating EAU.
CONCLUSIONS
Our results demonstrate that PTX plays a key role as a virulence factor in initiating autoimmune inflammation via DCs by inhibiting Wnt/β-catenin signaling in EAU, and highlight the potential mechanism by which infection can trigger apparent autoimmunity.
Topics: Mice; Animals; Pertussis Toxin; Autoimmunity; Wnt Signaling Pathway; beta Catenin; Autoimmune Diseases; Uveitis; Inflammation; Dendritic Cells
PubMed: 36739434
DOI: 10.1186/s12974-023-02707-y