scholarly journals Prophylactic potential of cytolethal distending toxin B (CdtB) subunit of typhoid toxin against Typhoid fever

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Reena Thakur ◽  
Preeti Pathania ◽  
Navneet Kaur ◽  
Vattan Joshi ◽  
Kanthi Kiran Kondepudi ◽  
...  
Keyword(s):  
Typhoid Fever ◽  
Bacterial Load ◽  
Lethal Dose ◽  
Active Immunization ◽  
Effective Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Morphological Alterations ◽  
Log Reduction ◽  
Typhoid Toxin

AbstractTyphoid fever caused by Salmonella enterica serovar Typhi (S.Typhi) continues to be a major problem, especially in developing countries. Due to the rapid emergence of multi-drug-resistant (MDR) strains, which limits the efficacy of conventional antibiotics as well as problems associated with the existing vaccines, efforts are being made to develop effective prophylactic agents. CdtB subunit of typhoid toxin was selected for assessing its vaccine potential due to its high conservation throughout the Typhi strains. In-vitro assessment of DNase activity of cloned and purified CdtB protein showed a significant decrease in the band intensity of DNA. The measure of metabolic activity and morphological alterations assessed using different cell lines in the presence of CdtB protein showed no significant signs of toxicity. These observations were further strengthened by cell cycle analysis, assessed by flow cytometry. Keeping these observations in mind, the immunoprotective potential of CdtB was assessed using S.Typhi induced mouse peritonitis model. A significant titer of IgG antibodies (>128000) against CdtB protein was recorded in the immunized mice by enzyme-linked immunosorbent assay (ELISA), which was also validated by immunoblotting. Active immunization with the protein protected 75% mice against a lethal dose of S.Typhi Ty2. The data indicated a significant (up to 5 log) reduction in the bacterial load in the spleen and liver of immunized-infected mice compared to control (unimmunized-infected) mice which might have resulted in the modulation of histoarchitecture of spleen and liver and the levels of cytokines (IL-6, TNF-α and IL-10) production; thereby indicating the effectiveness of the subunit. The observations deduced from the study give the proof of concept of immunogenic potential of protein. However, further studies involving the immunoreactivity of CdtB with the statistically significant number of sera samples obtained from the human patients would be helpful in establishing the relevance of CdtB protein in humans and for making the strategies to develop it as an effective vaccine candidate.

scholarly journals Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 508 ◽  
Author(s):  
Daniela Luz ◽  
Maria Amaral ◽  
Flavia Sacerdoti ◽  
Alan Bernal ◽  
Wagner Quintilio ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Fab Fragment ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Morphological Alterations ◽  
Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

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 Bone marrow function. I. Peripheral T cells are responsible for the increased auto-antiidiotype response of older mice.

1985 ◽  
Vol 161 (5) ◽  
pp. 1237-1242 ◽  
Author(s):  
Y T Kim ◽  
E A Goidl ◽  
C Samarut ◽  
M E Weksler ◽  
G J Thorbecke ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Immune Function ◽  
Lethal Dose ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Peripheral T Cells ◽  
Bone Marrow Function ◽  
Low Levels

After immunization with trinitrophenyl (TNP)-Ficoll, mice produced both anti-TNP antibodies and auto-anti-idiotype (auto-anti-Id) antibodies specific for the anti-TNP antibody. Older animals produced more auto-anti-Id than did young animals. When mice were exposed to a normally lethal dose of irradiation while their bone marrow (BM) was partially shielded, they survived and slowly (6 wk) regained immune function, as indicated by the number of nucleated cells in their spleen and the in vitro primary plaque-forming cell (PFC) response of their spleen cells to TNP-treated aminoethylated polyacrylamide beads. Recovery is presumably the result of repopulation of the peripheral lymphoid system by cells originating in the BM. By enzyme-linked immunosorbent assay (ELISA), and by hapten-augmentable PFC assay, we show that, after recovery from irradiation with their BM shielded, old animals produce low auto-anti-Id responses, like those of young animals. The transfer of splenic T cells into mice irradiated with their BM shielded provided evidence that the magnitude of the auto-anti-Id response is controlled by the peripheral T cells. Thus, mice that received splenic T cells from aged donors produced high levels of auto-anti-Id while those that received splenic T cells from young donors produce low levels of auto-anti-Id.

scholarly journals Cocculus hirsutus-derived phytopharmaceutical drug has potent anti-dengue activity

2020 ◽  
Author(s):  
Ankur Poddar ◽  
Rahul Shukla ◽  
Hemalatha Beesetti ◽  
Upasana Arora ◽  
Ravi Kant Rajpoot ◽  
...  
Keyword(s):  
At Risk ◽  
Aqueous Extract ◽  
Aerial Part ◽  
Lethal Dose ◽  
Drug Substance ◽  
Health Concern ◽  
Effective Vaccine ◽  
Cocculus Hirsutus

AbstractBackgroundDengue is a serious public health concern worldwide, with ~3 billion people at risk of contracting dengue virus (DENV) infections. Currently, no effective vaccine or drug is available for the prevention or treatment of dengue, which leaves only anti-mosquito strategies to combat this disease. The present study was initiated to determine the in-vitro and in vivo protective effects of a plant-derived phytopharmaceutical drug for the treatment of dengue.Methodology/Principal FindingsIn our previous report, we had identified methanolic extract of the aerial parts of Cissampelos pareira to exhibit in vitro and in vivo anti-dengue activity against all the four DENV serotypes. In the current study, we have identified another Indian medicinal plant, Cocculus hirsutus, which has a more potent anti-dengue activity than C. pareira. The activity has been evaluated through flow-cytometry-based virus inhibition assay. Interestingly, the stem of C. hirsutus was found to be more potent than the aerial part irrespective of the extraction solvent used viz., denatured spirit, hydro-alcohol (50:50) and water. Hence, the aqueous extract of stem of C. hirsutus (AQCH) was further advanced for investigations because of greater regulatory acceptance. The AQCH exhibited dose-dependent inhibition of release of DENV and its secretory antigen, NS1. Five chemical markers viz. Sinococuline, 20-Hydroxyecdysone, Makisterone-A, Magnoflorine and Coniferyl alcohol were identified as the major chemical ingredients of the AQCH extract. These chemicals were subsequently used for extract standardisation. Importantly, AQCH completely protected AG129 mice at 25 mg/kg/dose body weight when fed 4 times a day post-infection with a lethal dose of DENV-2 S221 strain. Because of its potential as an effective phytopharmaceutical drug against dengue, AQCH, has been formulated into tablets for further pre-clinical and clinical developments.Conclusions/SignificanceWe provide evidence of the pan anti-dengue potential of C. hirsutus-based phytopharmaceutical drug as determined through in vitro and in vivo experiments. We have also characterized five chemical entities in the drug substance, which provides means for standardization of drug substance and drug product. Based on these findings, a program to develop a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue has been initiated.Author summaryThere is an urgent need to develop a safe and effective drug against dengue, which is a rapidly expanding mosquito-borne viral disease. Half of the world’s population has been estimated to be at risk of contracting this disease and the situation remains grim due to lack of an approved drug. We aimed to develop an ethnopharmacological drug against dengue by exploring traditional Indian medicinal science, Ayurveda. This led us to identify a creeper, Cocculus hirsutus, as a more potent anti-dengue plant than Cissampelos pareira, reported in our earlier published study. The stem part of C. hirsutus was found to be more efficacious in inhibiting the propagation of dengue viruses (DENVs) in cell culture than its aerial part. Hence, we chose to advance aqueous extract of stem of C. hirsutus (AQCH) for further studies. Importantly, AQCH also protected immune-compromised mice from lethal DENV infection, which is suggestive of its potential clinical relevance. We have identified five chemical marker compounds in AQCH to gauge the quality and consistency of extract preparation and its formulation into stable tablets. Based on the findings of this study, we have undertaken the development of a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue.

scholarly journals Candidate Vaccine against Botulinum Neurotoxin Serotype A Derived from a Venezuelan Equine Encephalitis Virus Vector System

2001 ◽  
Vol 69 (9) ◽  
pp. 5709-5715 ◽  
Author(s):  
John S. Lee ◽  
Peter Pushko ◽  
Michael D. Parker ◽  
Mark T. Dertzbaugh ◽  
Leonard A. Smith ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Lethal Dose ◽  
Candidate Vaccine ◽  
Vector System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Venezuelan Equine Encephalitis ◽  
Serotype A ◽  
Replicon Vector ◽  
Botulinum Neurotoxin Serotype A

ABSTRACT A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes andcis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (HC) of the BoNT/A heavy chain was cloned into the replicon vector (HC-replicon). Cotransfection of BHK cells in vitro with the HC-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, HC VEE replicon particles (HC-VRP). Cells infected with HC-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of HC-VRP, mice were vaccinated with various doses of HC-VRP at different intervals. Mice inoculated subcutaneously with HC-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

The Fraction of the Snake Venom, its Leishmanicidal Effect, and the Stim-ulation of an Anti-Leishmania Response in Infected Macrophages

2020 ◽  
Vol 20 ◽  
Author(s):  
Saeideh Nikpour ◽  
Fatemeh Tabatabaie ◽  
Iraj Sharifi ◽  
Mahshid Mostafavi ◽  
Razieh Tavakoli Oliaee ◽  
...  
Keyword(s):  
Snake Venom ◽  
High Performance ◽  
Inhibitory Effect ◽  
Control Measures ◽  
Effective Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Th2 Response ◽  
Wide Range

Background and aims:: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOV-F11) on L. infantum and its broad mode of action. Methods:: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue) and macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme-linked immunosorbent assay (ELISA) on L. infantum pro-mastigote and amastigote stages were used. IC50 values of L. infantum stages, CC50 value and apoptosis were also analyzed. Results:: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent man-ner compared to the untreated control group. Interleukin (IL)-12, TNF-α and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p ˂ 0.001). Reactive oxygen species (ROS) detection showed statistically a significant increase (p ˂ 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity which displayed a signif-icant reduction (p < 0.001). Conclusion:: NNOV-F11 possesses a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic–like mechanisms and inhibited L-ARG activity which collectively led to the parasite death. Further studies are crucial to assess the effect of the fraction NNOV-F11 on animal models or clinical settings.

scholarly journals A Single Intramuscular Injection of Recombinant Plasmid DNA Induces Protective Immunity and Prevents Japanese Encephalitis in Mice

2000 ◽  
Vol 74 (9) ◽  
pp. 4244-4252 ◽  
Author(s):  
Gwong-Jen J. Chang ◽  
Ann R. Hunt ◽  
Brent Davis
Keyword(s):  
Dna Vaccine ◽  
Japanese Encephalitis ◽  
Plasmid Dna ◽  
Recombinant Plasmid ◽  
Culture Media ◽  
Early Gene ◽  
Effective Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Old Mice

ABSTRACT Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50,000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.

In vitro pharmacodynamics of omadacycline against Escherichia coli and Acinetobacter baumannii

2020 ◽  
Author(s):  
A R Noel ◽  
M Attwood ◽  
K E Bowker ◽  
A P MacGowan
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Pharmacokinetic Model ◽  
Bacterial Load ◽  
Gram Negative ◽  
E Coli ◽  
Log Reduction ◽  
Static Effect

Abstract Background The pharmacodynamics of omadacycline have been extensively studied against Gram-positive pathogens but less information is available for Gram-negative pathogens. We describe the pre-clinical pharmacodynamics of omadacycline against Escherichia coli and Acinetobacter baumannii. Methods An in vitro dilutional pharmacokinetic model was used. Exposure experiments with fAUC/MIC ratios ranging from 0 to 1200 were performed using five strains of E. coli and five strains of A. baumannii. Reduction in bacterial load and changes in population profiles were measured. Results The fAUC/MIC targets against E. coli for 24 h static and −1 log reduction in load were 25.3 ± 17.2 and 42.7 ± 32.5, respectively. For A. baumannii the fAUC/MIC for 24 h static effect was 108.1 ± 38.6. Changes in population profiles were observed for E. coli at fAUC/MIC ratios of ≤200 and for A. baumannii up to 1200. MICs were increased 2–32 fold. Conclusions fAUC/MIC targets for A. baumannii are greater than for E.coli and changes in population profiles more likely. E. coli fAUC/MIC targets align with in vivo data and will be useful in determining omadacycline dosing for this pathogen.

Neutralization of typhoid toxin by alpaca-derived, single-domain antibodies targeting the PltB and CdtB subunits

2021 ◽  
Author(s):  
Hari P. Dulal ◽  
David J. Vance ◽  
Durga P. Neupane ◽  
Xiangcheng Chen ◽  
Jacqueline M. Tremblay ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Bacterial Pathogen ◽  
Salmonella Typhi ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Function Defect ◽  
Single Domain Antibodies ◽  
Typhoid Toxin

Typhoid toxin is secreted by the typhoid fever-causing bacterial pathogen Salmonella Typhi and has tropism for immune cells and brain endothelial cells. Here, we generated a camelid single domain antibody (VHH) library from typhoid toxoid-immunized alpacas and identified 41 VHHs selected on the glycan-receptor binding PltB and nuclease CdtB. VHHs exhibiting potent in vitro neutralizing activities from each sequence-based family were epitope binned via competition ELISAs, leading to 6 distinct VHHs, two anti-PltB (T2E7 and T2G9) and four anti-CdtB VHHs (T4C4, T4C12, T4E5, and T4E8), whose in vivo neutralizing activities and associated toxin neutralizing mechanisms were investigated. We found that T2E7, T2G9, and T4E5 effectively neutralized typhoid toxin in vivo , as demonstrated by 100% survival of mice administered a lethal-dose of typhoid toxin and with little to no typhoid toxin-mediated upper motor function defect. Cumulatively, these results highlight the potential of the compact antibodies to neutralize typhoid toxin by targeting the glycan-binding and/or nuclease subunits.

scholarly journals Differential Selection of Multidrug Efflux Mutants by Trovafloxacin and Ciprofloxacin in an Experimental Model ofPseudomonas aeruginosa Acute Pneumonia in Rats

2001 ◽  
Vol 45 (2) ◽  
pp. 571-576 ◽  
Author(s):  
O. F. Join-Lambert ◽  
M. Michéa-Hamzehpour ◽  
T. Köhler ◽  
F. Chau ◽  
F. Faurisson ◽  
...  
Keyword(s):  
Bacterial Load ◽  
Resistant Bacteria ◽  
Multidrug Efflux ◽  
Time Curve ◽  
Treatment Regimens ◽  
Log Reduction ◽  
Dosing Regimens ◽  
Selection Of

ABSTRACT The ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo was studied in a model of acute Pseudomonas aeruginosa pneumonia in rats. Twelve hours after intratracheal inoculation of 106 CFU of P. aeruginosastrain PAO1 enmeshed in agar beads, two groups of 12 rats were treated by three intraperitoneal injections of each antibiotic given every 5 h. Dosing regimens were chosen to obtain a comparable area under the concentration-time curve from 0 to infinity/MIC ratio of 27.9 min for trovafloxacin (75 mg/kg of body weight) and of 32.6 min for ciprofloxacin (12.5 mg/kg). Twelve rats were left untreated and served as controls. Rats were sacrificed 12 h after the last injection (34 h after infection) for lung bacteriological studies. Selection of resistant bacteria was determined by plating lung homogenates on Trypticase soy agar plates containing antibiotic. In untreated animals, the frequency of resistant colonies was 10-fold higher than in agar beads. Compared to controls, both treatment regimens resulted in a 2-log reduction of lung bacterial load. The frequency of resistant colonies was 10-fold less with trovafloxacin than with ciprofloxacin at twice the MIC (7.4 × 10−5 versus 8.4 × 10−4, respectively) (P < 0.05) and at four times the MIC (6.2 × 10−4 versus 5.0 × 10−5, respectively) (P < 0.05). A multidrug resistance phenotype typical of efflux mutants was observed in all 41 randomly tested colonies obtained from treated and untreated rats. In agreement with in vitro results, trovafloxacin and ciprofloxacin preferentially selected MexCD-OprJ and MexEF-OprN overproducers, respectively. These results demonstrate the differential ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo and highlight the rapid emergence of those mutants, even without treatment.

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