Search

Whilst various remedial human monoclonal antibodies have been developed to treat the potentially life-threatening systemic complications associated with anthrax infection, an optimal and universally effective administration route has yet to be established. In the later stages of infection when antibody administration by injection is more likely to fail one possible route to improve outcome is via the use of an antibody-bound, adsorbent haemoperfusion device. We report here the development of an adsorbent macroporous polymer column containing immobilised B. anthracis exotoxin-specific antibodies, PANG (a non-glycosylated, version of a plant-produced human monoclonal antibody) and Valortim (a fully human monoclonal N-linked glycosylated antibody), for removal of anthrax protective antigen (PA) from freshly frozen human plasma and human whole blood. In addition, we have demonstrated that continuous extracorporeal blood recirculation through a Valortim-bound haemoperfusion column significantly reduced the blood plasma concentration of anthrax PA over 2 hours using an in vivo PA rat infusion model. This work provides proof-of-concept evidence to support the development of such alternative detoxification platforms.

Tuberculosis is a major global health hazard. The search for new antimycobacterials has focused on such as screening combinational chemistry libraries or designing chemicals to target predefined pockets of essential bacterial proteins. The relative ineffectiveness of these has led to a reappraisal of natural products for new antimycobacterial drug leads. However, progress has been limited, we suggest through a failure in many cases to define the drug target and optimize the hits using this information. We highlight methods of target discovery needed to develop a drug into a candidate for clinical trials. We incorporate these into suggested analysis pipelines which could inform the research strategies to accelerate the development of new drug leads from natural products.

Clostridium difficile is the primary cause of healthcare associated diarrhoea globally and produces spores which are resistant to commonly used biocides and are able persist on contaminated surfaces for months. This study examined the effect of sublethal concentrations of the biocide sodium dichloroisocyanurate (NaDCC) on the viability of spores produced by 21 clinical isolates of C. difficile representing a range of PCR ribotypes. Spores exposed to 500 ppm NaDCC for 10 minutes exhibited between a 4 – 6 log10 reduction in viability which was independent of spore PCR ribotype. The effect of sublethal concentrations of biocide on the surface properties of exosporium positive and negative clinical isolates was determined using a spore adhesion to hydrocarbon assay. These isolates differed markedly in their responses suggesting that exposure to biocide can have a profound effect on hydrophobicity and thus the ability of spores to adhere to surfaces. This raises the intriguing possibility that sublethal exposure to NaDCC could inadvertently promote the spread of the pathogen in healthcare facilities.

Hostile reconnaissance is vital to successful terrorist activity. Individuals carrying out this activity are likely to experience raised levels of stress and this will manifest itself at biological, physiological, psychological and behavioural levels, providing an opportunity for detection. A field trial was undertaken in an ecologically valid environment measuring variables considered likely to be salient during hostile intent. The parameters examined in the field trial varied in a predictable manner and suggest that stressed individuals secrete a volatile steroid based marker that could form the basis for remote detection. Thus, overall the findings of this research provide a validated model of hostile intent that can be used by other researchers to test interventions aimed at detecting or deterring hostile intent.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC50 for RT was 0.08 ± 0.004 ng/mL whereas the IC50 for RT + 100 μM eGCG was 3.02 ± 0.572 ng/mL, indicating that eGCG mediated a significant (p

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy forBacillus anthracisinfection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] ofB. anthracisSterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected withB. anthracisSterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim®), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim® attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim® antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p

Individuals trying to conceal knowledge from interrogators are likely to experience raised\ud levels of stress that can manifest itself across biological, physiological, psychological and\ud behavioural factors, providing an opportunity for detection. Using established research\ud paradigms an innovative scalable interrogation was designed in which participants were\ud given a ‘token’ that represented information they had to conceal from interviewers. A control\ud group did not receive a token and therefore did not have to deceive the investigators. The\ud aim of this investigation was to examine differences between deceivers and truth-tellers\ud across the four factors by collecting data for cortisol levels, sweat samples, heart-rate,\ud respiration, skin temperature, subjective stress ratings and video and audio recordings. The\ud results provided an integrated understanding of responses to interrogation by those actively\ud concealing information and those acting innocently. Of particular importance, the results also\ud suggest, for the first time in an interrogation setting, that stressed individuals may secrete a\ud volatile steroid based marker that could be used for stand-off detection. The findings are\ud discussed in relation to developing a scalable interrogation protocol for future research in this\ud area.

The unpredictable nature of bio-terrorism compels us to develop medical countermeasures that will enable authorities to treat individuals exposed to agents such as anthrax. We report the feasibility of producing a protective, human-derived, monoclonal antibody directed against the protective antigen (PA) of Bacillus anthracis in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting antibody was able to neutralize toxin activity in vitro and in vivo at a comparable level to that seen for its hybridoma-produced counterpart.

DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development. DNA vaccination against anthrax is an active area of research showing promising results at present, which in the short-term and in the future could form the basis for new advances in multi-agent vaccine development. The anthrax 'model' constitutes an important experimental system for genetic immunisation technology development.

Anthrax is a disease caused by a Gram-positive, aerobic, spore-forming bacillus called Bacillus anthracis. This bacterium primarily infects animals and also humans, sometimes with fatal consequence...

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile.

The sequencing of the Bacillus anthracis genome and virulence plasmids represents the greatest advance in anthrax research in the past 100 years. The data will provide the foundation of all future work on this organism and will be invaluable to researchers in their battle to understand the basis of the host-microbe interaction.

Bacillus anthracis is a pathogen of animals which rarely infects humans. Its use as a bioweapon has stimulated efforts to develop genetic typing methods and therapeutics to respond to an attack. Of particular concern is the transfer of virulence genes from B. anthracis to other closely related strains of bacillus.

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species.

Protective antigen (PA), the major protective component of the existing vaccine, is a potent immunogen. Protective antigen in alhydrogel induced a high serum IgG titre (> log10 4) in both the C57B16 and Balb/c mouse and the predominant subclass of antibody induced was IgG1, indicating that the response to PA was predominantly Th2 directed. When plasmid DNA encoding PA was used to immunize the Balb/c mouse, a low serum IgG titre was detected (≤log10 1), which was slightly increased by boosting with plasmid DNA. However, when mice immunized with plasmid DNA were later boosted with rPA, a significant and rapid increase in titre (up to threefold) was observed. Priming mice with PA-encoding plasmid DNA may be a mechanism of enhancing and accelerating the immune response to PA.

The IgG anti-protective antigen subclass antibody response of individuals who had been infected with anthrax was compared with that of healthy individuals immunized with the UK licensed anthrax vaccine. The predominant subclass in both groups was IgG1. In addition, IgG3 was seen in convalescent serum while vaccinees produced IgG2, IgG3 and IgG4 subclass. The significance of these results is discussed. Further work is required to determine the role of antibodies in mediating protective immunity in man.

Aim: Anthrax is caused by the bacterium Bacillus anthracis. Although primarily a disease of animals, it can also infect man, sometimes with fatal consequences. As a result of concerns over the illicit use of this organism, considerable effort is focussed on the development of therapies capable of conferring protection against anthrax. This brief review will describe the efforts being made to address these issues. Methods and Results: A review of the literature and the proceedings of the sixth international conference on anthrax, held in Santa Fe, USA in 2005 shows intense activity, but there has been as yet no real progress. While effective antibiotics, antitoxins and vaccines are available, concerns over their toxicity and the emergence of resistant strains have driven the development of second-generation products. The principal target for vaccine development is Protective Antigen (PA), the nontoxic cell-binding component of anthrax lethal toxin. While the recombinant products currently undergoing human clinical trials will offer considerable advantages in terms of reduced side effects and ease of production, they would still require multiple, needle-based dosing, and the inclusion of the adjuvant alum makes them expensive to administer and stockpile. To address these issues, researchers are developing vaccine formulations, which stimulate rapid protection following needle-free injection (nasal, oral or transcutaneous), and are stable at room temperature to facilitate stockpiling and mass vaccination programs. Conclusions: An array of medical countermeasures targeting B. anthracis will become available over the next 5–10 years. Significance and Impact of the Study: The huge investment of research dollars is expected to dramatically expand the knowledge base. A better understanding of basic issues, such as survival in nature and pathogenesis in humans, will facilitate the development of new modalities to eliminate the threat posed by this organism.

Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (gerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN- )o r opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN- and IgG was no better than either treatment alone. Under no condition was there killing of gerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium) and sonicated (exosporium) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFNincreased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages. Bacillus anthracis, the causative agent of anthrax, is a highly virulent gram-positive and spore-forming bacterium that is typically acquired through contact with anthrax-infected animals or animal products or atypically through intentional exposure as a biological weapon (6, 8). Virulent strains of B. anthracis carry two large plasmids, pXO1 and pXO2, that carry the genes encoding anthrax toxin production and capsule formation, respectively. Dormant spores are highly resistant to adverse environmental conditions but are able to reestablish vegetative growth in the presence of favorable environmental

The anthrax bipartite lethal toxin (protective antigen (PA) and lethal factor (LF))-specific antibody responses of humans receiving the UK licensed anthrax vaccine were determined. The PA-specific IgG response peaked two weeks post immunization and fell back to pre-boost levels by week 12. The heterogeneity of the host population modulated the extent of the PA-specific antibody response. Significantly lower levels of LF-specific antibodies were also detected. Vaccinated individuals recognized the same PA epitope as the protective mouse lethal toxin neutralizing monoclonal 2D3 suggesting that this may also be a target for human protection.

Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly- l -lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10 3 median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

Recombinant protective antigen (rPA), expressed by Bacillus subtilis WB600 (pPA101), has been purified to homogeneity and the protective efficacy against a Bacillus anthracis challenge has been investigated. rPA was fractionated from culture supernatant fluid by ammonium sulphate, followed by anion exchange chromatography using DEAE Streamline™, anion-exchange chromatography on FPLC MonoQ HR 10/10 and finally, gel filtration chromatography on FPLC Superose 12 HR 10/30, to yield 7 mg rPA per litre of culture. The protective efficacy of rPA against an airborne challenge with the AMES strain of B. anthracis was determined in the presence of the adjuvants, alhydrogel and Ribi, and compared to that achieved by the current UK human vaccine in guinea pigs. rPA combined with the Ribi adjuvant was found to provide 100% protection against challenge.

Vancomycin resistant enterococci are increasingly being isolated from inpatients. This report describes the colonial variation present in most isolates of vancomycin resistant Enterococcus faecium obtained at this hospital. Colonial variants within the same culture were indistinguishable by antimicrobial susceptibility, biochemical reactions, and ribotyping. Failure to appreciate this colonial variation will lead to pure cultures being regarded as contaminated or mixed.

Seventy-four of 155 catheter specimens of urine yielded organisms resistant to chlorhexidine. The inhibitory effect of urine against chlorhexidine is reported.