Monoclonal antibodies as tools in antigen detection assay and vaccine development : design of a sensitive detection test for Brucella bacteria and profiling of the malaria vaccine candidate antigen reticulocyte-binding homolog 2 (PfRH2)

We aimed at identifying immunodominant Brucella antigens for implementation in new detection tools or for subunit vaccine development. In particular, our strategy was to produce Brucella cell surface antigen-specific monoclonal antibodies (mAbs) for the development of an antigen capture assay for the detection of Brucella cells as potential bio threat agents in complex samples. We generated a panel of Brucella lipopolysaccharide (LPS)-specific mAbs by immunising mice with inactivated B. melitensis and B. abortus cells. The mAbs recognised Brucella species with ‘smooth’ LPS independently of the way how the bacterial cells were inactivated. Two mAbs were implemented into a bead-based Luminex assay detecting ‘smooth’ Brucella spp. with species-dependent detection limits of 2 x 102 to 8 x 104 cells per mL. Integration of the Luminex assay into a multiplex format enabled simultaneous detection of Brucella spp. and three other bio threat agents within a single sample. The developed Luminex assay may be applied for the detection of whole Brucella cells both in natural Brucella outbreak and in bioterrorism attack scenarios. We also tried to generate mAbs against Brucella cell surface proteins from mice immunised with inactivated whole Brucella cells. While serum antibody responses against both LPS and protein antigens were seen in Western blotting analyses, attempts to generate protein-specific mAbs failed, most likely due to the immunodominant nature of the LPS. Western blot analyses with Brucella lysate also identified antibodies against some immunodominant Brucella proteins in the serum of cattle naturally infected with Brucella spp., however, identification of the recognised proteins with a Brucella-specific peptide microarray failed. In a second part of the thesis we aimed at evaluating the potential of the Plasmodium falciparum reticulocyte-binding homolog 2 (PfRH2) present in the rhoptries as a malaria blood stage vaccine antigen. We produced PfRH2-specific mAbs by immunising mice with the 40kDa receptor-binding domain of PfRH2. The PfRH2-specific mAbs cross-reacted with the natural PfRH2 protein present in schizont stage parasites and showed a rhoptry-characteristic staining pattern in immunofluorescence microscopy. However when evaluated in functional in vitro and in vivo assays PfRH2 specific mAbs showed no inhibitory effect on erythrocyte invasion. Furthermore, the invasion-inhibitory effect of mAbs specific for the cysteine-rich protective antigen (PfCyRPA) was not enhanced by PfRH2-specific mAbs

Salivary biomarkers in the context of gingival inflammation in children with cystic fibrosis

Abstract Background Cystic fibrosis (CF) is a life-threatening chronic inflammatory disease in children due to respiratory complications. Saliva could serve as reservoir of bacterial colonization and potentially reflect systemic inflammation. This study investigated whether salivary triggering receptor expressed on myeloid cells 1 (TREM-1), peptidoglycan recognition protein 1 (PGLYRP1), interleukin (IL)-1? and calprotectin are associated with CF or reflect concomitant gingival inflammation. Methods Ten CF (age:3-12yrs) and ten systemically healthy age-and-gender-matched children (C) were enrolled in the study. Individuals with CF underwent routine laboratory determinations. Probing pocket depth (PPD), gingival index (GI), plaque index (PI) and bleeding on probing (BOP) were recorded on fully erupted teeth and saliva samples collected. Salivary TREM-1, PGLYRP1, IL-1? and calprotectin were analysed by ELISA. Results Children with CF had significantly higher BOP scores (P = 0.001) and calprotectin levels (P = 0.017) compared to the C group. TREM-1, PGLYRP1 and IL-1? could not distinguish between CF and SH but showed positive correlation with GI, PI and BOP in both groups. Calprotectin levels positively correlated with procalcitonin (P = 0.014), thrombocyte counts (P = 0.001), mean platelet volume (P = 0.030) and with PGLYRP1 (P = 0.019) and IL-1? (P = 0.013) in CF children. Receiver operating characteristic curve analysis for calprotectin (CFvsC) showed an area under the curve of 0.79 (95% CI 0.58-0.99, P = 0.034). Conclusions CF children presented with higher gingival inflammation scores and salivary calprotectin levels, that correlated with systemic inflammatory markers. Salivary calprotectin levels were not associated with periodontal parameters. Hence, preliminary data demonstrate that salivary calprotectin might have a chairside diagnostic potential for CF in children. This article is protected by copyright. All rights reservedPeer reviewe

A point-of-care test of active matrix metalloproteinase-8 predicts triggering receptor expressed on myeloid cells-1 levels in saliva

Background This cross-sectional study aims to investigate if a point-of-care (PoC) test of active matrix metalloproteinase-8 (aMMP-8) predicts levels of inflammation amplifier triggering receptor expressed on myeloid cells-1 (TREM-1) and its putative ligand the neutrophil peptidoglycan recognition protein 1 (PGLYRP1) in saliva. Methods Forty-seven adolescents, aged 15 to 17 years, were tested with aMMP-8 PoC test, which was followed by a full-mouth clinical examination of the assessment of periodontal, mucosal, and oral health. TREM-1 and PGLYRP1 levels were analyzed by ELISA. The immunofluorometric assay (IFMA) specific for aMMP-8 was used as the reference method. Results Fourteen saliva samples out of a total of 47 showed positivity for aMMP-8 PoC test. Both the TREM-1 and the aMMP-8 (IFMA) levels were significantly elevated among the aMMP-8 PoC test positives compared with the PoC test negatives (P = 4 mm was significantly lower among the adolescents that had a negative aMMP-8 PoC test result, and TREM-1 levels = 4 mm (P <0.001). Conclusion The present study validated usability of aMMP-8 PoC test for predicting "proinflammatory" salivary profile and periodontal health status in adolescents.Peer reviewe

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation

OBJECTIVE: Salivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model. METHODS: Dental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18). RESULTS: The TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, 'fast' responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than 'slow' responders, who showed a lagged inflammatory response. CONCLUSION: The results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases

Active matrix metalloproteinase-8 and interleukin-6 detect periodontal degeneration caused by radiotherapy of head and neck cancer: a pilot study

Background: This cohort study investigated the role of the active matrix metalloproteinase-8 (aMMP-8) and interleukin-6 (IL-6) as oral fluid biomarkers for monitoring the periodontal degeneration occurring in head and neck cancer (HNC) patients treated by radiotherapy. Research design and methods: Eleven patients, aged 28-74, diagnosed with HNC were included in the study. Complete periodontal and oral examinations were performed pre-radiotherapy and 1 month after radiotherapy. Mouthrinse samples (pre-radiotherapy, after 6 weeks of radiotherapy and 1 month after radiotherapy) were assayed by aMMP-8 point-of-care-kit (PerioSafe®/ORALyzer®) for aMMP-8 and ELISA for IL-6.Results: HNC radiotherapy had a deteriorating impact on the periodontium and a significant impact on periodontal biomarkers aMMP-8 and IL-6 and increased their levels in mouthrinse. Clinical-attachment-loss (CAL) (site of greatest loss: mean = 1.7 mm, range = 1-3 mm) corresponding to rapid progression of periodontitis. There was a positive repeated measures correlation (rmcorr = 0.667) between the aMMP-8 and IL-6 levels. Conclusions: Elevated aMMP-8 levels were observed 1 month after radiotherapy among some HNC patients suggesting a prolonged increased susceptibility to further periodontal tissue destruction. Currently available aMMP-8 point-of-care testing could be useful to monitor and assess quantitatively online and real-time the risk of deterioration of periodontal health during HNC radiotherapy.</p

In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections

Soil-transmitted helminthiases affect more than one billion people among the most vulnerable populations in developing countries. Currently, control of these infections primarily relies on chemotherapy. Only five drugs are available, all of which have been in use for decades. None of the drugs are efficacious using single doses against all soil-transmitted helminths (STH) species and show low efficacy observed against Trichuris trichiura. In addition, the limited availability of current drug treatments poses a precarious situation should drug resistance occur. Therefore, there is great interest to develop novel drugs against infections with STH. Monepantel, which belongs to a new class of veterinary anthelmintics, the amino-acetonitrile derivatives, might be a potential drug candidate in humans. It has been extensively tested against livestock nematodes, and was found highly efficacious and safe for animals. Here we describe the in vitro and in vivo effect of monepantel, on Ancylostoma ceylanicum, Necator americanus, Trichuris muris, Strongyloides ratti, and Ascaris suum, five parasite-rodent models of relevance to human STH. Since we observed that monepantel showed only high activity on one of the hookworm species and lacked activity on the other parasites tested we cannot recommend the drug as a development candidate for human soil-transmitted helminthiases

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay.; To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed.; MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2 × 10(2) to 8 × 10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample.; Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations

Exploration of novel in vitro assays to study drugs against Trichuris spp

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100, 50mug/ml) for 72h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cos

Effects of monepantel, albendazole, levamisole, and pyrantel pamoate on <i>T. muris in vivo</i>.

<p>SD = standard deviation.</p>§<p>Mann-Whitney U test comparing the median of the worm burdens of control and treated mice.</p

50% inhibitory concentrations of monepantel, albendazole, levamisole, and pyrantel pamoate on <i>A. ceylanicum</i>, <i>N. americanus</i>, and <i>T. muris</i>.

<p>IC₅₀s (µg/ml) were calculated for monepantel, albendazole, levamisole, and pyrantel pamoate after 72 h on L3 and adult stages of <i>A. ceylanicum</i>, <i>N. americanus</i>, and <i>T. muris</i>. r = linear correlation coefficient of the median-effect plot, indicating the goodness of fit. r≥0.85 indicates a satisfactory fit. n.d.: not determined, fitting not possible.</p