Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Biophysical Characterization and Thermal Stability of Pneumococcal Histidine Triad Protein D in the Presence of Zinc and Manganese.

The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn2+ and Mn2+ were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn2+ and Mn2+. UV spectroscopy and fluorescence quenching studies showed significant Zn2+-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn2+ but revealed a Zn2+:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn2+. Only Zn+2, but not Mn+2, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.

Effective Targeted Photothermal Ablation of Multidrug Resistant Bacteria and Their Biofilms with NIR-Absorbing Gold Nanocrosses.

With the rapid evolution of antibiotic resistance in bacteria, antibiotic-resistant bacteria (in particular, multidrug-resistant bacteria) and their biofilms have been becoming more and more difficult to be effectively treated with conventional antibiotics. As such, there is a great demand to develop a nonantibiotic approach in efficiently eliminating such bacteria. Here, multibranched gold nanocrosses with strong near-infrared absorption falling in the biological window, which heat up quickly under near-infrared-light irradiation are presented. The gold nanocrosses are conjugated to secondary and primary antibodies for targeting PcrV, a type III secretion protein, which is uniquely expressed on the bacteria superbug, Pseudomonas aeruginosa. The conjugated gold nanocrosses are capable of completely destroying P. aeruginosa and its biofilms upon near-infrared-light irradiation for 5 min with an 800 nm laser at a low power density of ≈3.0 W cm(-2) . No bacterial activity is detected after 48 h postirradiation, which indicates that the heat generated from the irradiated plasmonic gold nanocrosses attached to bacteria is effective in eliminating and preventing the re-growth of the bacteria. Overall, the conjugated gold nanocrosses allow targeted and effective photothermal ablation of multidrug-resistant bacteria and their biofilms in the localized region with reduced nonspecific damage to normal tissue.

Immunisation of mares with binding domains of toxins A and B of Clostridium difficile elicits serum and colostral antibodies that block toxin binding.

REASONS FOR PERFORMING STUDY: Enterocolitis caused by Clostridium difficile (C. difficile) is a serious, sometimes fatal, disease of neonatal foals and older horses. Toxins A and B (TcdA and B) produced by C. difficile are important virulence factors. Immunisation of mares with receptor binding domains of toxins may prevent or reduce the severity of C. difficile colitis in foals. OBJECTIVES: To determine whether antibodies generated in the pregnant mare to the binding regions of TcdA and B will neutralise TcdA and B toxicity. METHODS: Sequences encoding the binding domains of each toxin were isolated by PCR amplification from C. difficile JF09, a foal isolate, and cloned and expressed into pET15b. Thirteen mares were immunised twice 2 weeks apart with 200 µg of each recombinant protein with Quil A 2 months prior to foaling. Antibodies were assayed in the sera and colostrum by ELISA and for ability to block the cytopathic activity of each of toxin for equine endothelial cells. RESULTS: All mares produced strong serum antibody responses to the binding domain of each toxin. A high level of toxin-specific antibodies was also detected in colostrum and in most foal sera 2 days after suckling. Diluted sera and colostrum premixed with either TcdA or B had no effect on the morphology of equine endothelial cells. Application of the same concentration of toxins alone or premixed with nonimmune mare/foal serum or colostrum led to an unambiguous cytopathic effect that ranged from complete degradation to varying degrees of cell rounding. CONCLUSIONS: Immunisation of pregnant mares with recombinant binding domains of TcdA and B of C. difficile resulted in the production of specific antibodies in serum and colostrum that blocked the cytopathic activity of toxins. POTENTIAL RELEVANCE: Results of studies support the feasibility of a prepartum vaccine against C. difficile enterocolitis in foals.

Cross reaction between proteins from Larrea divaricata Cav. (jarilla) and cellular and extracellular proteins of Pseudomonas aeruginosa.

Larrea divaricata is widely used in folk medicine to treat different pathologies, but little is known about its immunological properties. Pseudomonas aeruginosa is an opportunistic pathogen which causes several intrahospitalary infections. We aimed to assess the immunological relation between proteins from a crude extract of L. divaricata Cav. (JPCE) and cellular and extracellular proteins (EP) of P. aeruginosa, as well as to establish the cross reactivity between proteins of both species using a mouse anti-JPCE serum. Protein profiles of JPCE and P. aeruginosa were analyzed by SDS-PAGE. The percentage of similarity of protein bands between these two species was 43-57%. However, JPCE proteins were immunogenic. The reactivity of mouse anti-JPCE antibodies against different fractions was studied by western blot. The anti-JPCE serum detected several antigenic bands on different bacterial proteins. Several common immunoreactive bands were detected (27-100%) when bacterial proteins were incubated with anti-JPCE serum (heterologous reaction) and anti-bacterial proteins serum (homologous reaction). By enzyme-linked immunosorbant assay (ELISA) assays, high titers of anti-JPCE against different types of cellular bacterial fractions were observed (1/1280-1/2080). Our data clearly demonstrate that antibodies elicited with L. divaricata crude extract are able to cross-react with cellular and EP of P.aeruginosa. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P.aeruginosa.

Targeting type III secretion in Yersinia pestis.

Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

An approach to obtain specific polyclonal antisera to Xanthomonas campestris pv. cyamopsidis and its potential application in indexing of infected seeds of guar.

Clusterbean seed health testing is warranted since the pathogen (Xanthomonas campestris pv. cyamopsidis (Xccy)) is seed-borne and seed-transmitted. A polyclonal antibody was developed in rabbit via subcutaneous and intramuscular injections and characterized for sensitivity, specificity and its applicability to ELISA which: (i) was sensitive in detecting as few as 102 cells ml - 1 at a titre of 1: 4000; (ii) was specific, since it reacted only with Xccy and not with other xanthomonads; (iii) reacted both with Xccy cells and culture filtrate, indicating that the antigenic determinant is a secretory component; (iv) was applicable and reliable in seed health testing since it reacted only with infected seeds and plant materials and not with healthy seeds and (v) a purified fraction of antibody was virulent-specific since heat-denatured and avirulent isolates were not detected. The ELISA thus developed is highly reproducible and therefore suitable for the evaluation of the potential disease status of seeds and plant health, which is appropriate for routine seed health testing.

A method for titration of inhibiting antibodies to bacterial immunoglobulin A1 proteases in human serum and secretions.

Bacterial IgA1 proteases specifically cleave IgA1, including S-IgA1, molecules into Fab alpha and Fc alpha fragments. Hereby these enzymes interfere with the protective functions of antibodies belonging to this isotype. Antibodies inhibiting IgA1 proteases have been detected in humans, but the titration of such antibodies is a matter of methodological concern. Because human serum and secretions contain IgA1 substrate, it is impossible to provide uniform substrate conditions for samples of IgA1 protease incubated with inhibitors differing in their origin and state of dilution. This study demonstrates that such variations in substrate are not prohibitive for a reliable titration of inhibiting antibodies. This was evident from experiments demonstrating that the variations do not interfere with the quantification of residual IgA1 protease activity provided the activity is measured in terms of the proportion of IgA1 substrate cleaved during incubation. Proportions of cleaved IgA1 were measured by exploiting the differential reactivity of cleaved and intact IgA1 molecules in an ELISA using anti-Fc alpha and enzyme-conjugated anti-light chain antibodies for catching and development, respectively. A protocol for the titration of IgA1 protease-inhibiting antibodies based on this ELISA is described. By application of the protocol to chromatographic fractions of saliva, IgA1 protease-inhibiting activity was found to co-purify with salivary S-IgA.