Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.

Assessment of CD200R Activation in Combination with Doxycycline in a Model of Melioidosis.

Antimicrobial resistance continues to be a global issue. Pathogens, such as Burkholderia pseudomallei, have evolved mechanisms to efflux certain antibiotics and manipulate the host response. New treatment strategies are therefore required, such as a layered defense approach. Here, we demonstrate, using biosafety level 2 (BSL-2) and BSL-3 in vivo murine models, that combining the antibiotic doxycycline with an immunomodulatory drug that targets the CD200 axis is superior to antibiotic treatment in combination with an isotype control. CD200-Fc treatment alone significantly reduces bacterial burden in lung tissue in both the BSL-2 and BSL-3 models. When CD200-Fc treatment is combined with doxycycline to treat the acute BSL-3 model of melioidosis, there is a 50% increase in survival compared with relevant controls. This benefit is not due to increasing the area under the concentration-time curve (AUC) of the antibiotic, suggesting the immunomodulatory nature of CD200-Fc treatment is playing an important role by potentially controlling the overactive immune response seen with many lethal bacterial infections. IMPORTANCE Traditional treatments for infectious disease have focused on the use of antimicrobial compounds (e.g. antibiotics) that target the infecting organism. However, timely diagnosis and administration of antibiotics remain crucial to ensure efficacy of these treatments especially for the highly virulent biothreat organisms. The need for early antibiotic treatment, combined with the increasing emergence of antibiotic resistant bacteria, means that new therapeutic strategies are required for organisms that cause rapid, acute infections. Here, we show that a layered defense approach, where an immunomodulatory compound is combined with an antibiotic, is better than an antibiotic combined with a relevant isotype control following infection with the biothreat agent Burkholderia pseudomallei. This approach has the potential to be truly broad spectrum and since the strategy includes manipulation of the host response it's application could be used in the treatment of a wide range of diseases.

Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

Dual route vaccination for plague with emergency use applications.

Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 × 104 median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 °C for 29 weeks, and 40 °C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed. In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.

Specific and charge interactions mediate collagen recognition by oral lactobacilli.

The mechanisms by which oral lactobacilli, one of the three major genera of cariogenic bacteria, attach to tooth surfaces are unknown. We hypothesize that recognition of collagen, the major component of dentin, may be a mechanism which localizes these bacteria to exposed root surfaces as well as to carious lesions which have penetrated the dentin. We found that the majority of oral Lactobacillus spp. strains recognize and bind collagen type I. Binding of 125I-labeled collagen type I to two strains of L. casei rhamnosus has been characterized in some detail. These strains were previously characterized with respect to their attachment to dentin (Switalski and Butcher, 1994). The process of 125I-collagen binding was mediated via specific as well as charge interactions. The putative adhesin-mediated (specific) interaction involved a limited number of bacterial surface components (2 x 10(3)/cell). Under conditions conducive for non-specific interactions (low ionic strength), the binding was higher by an order of magnitude. Collagen binding strains were found to adhere to collagen-coated surfaces, while strains unable to bind collagen adhered to a much lesser extent. Adherence of bacteria to collagen-coated surfaces could be competitively inhibited with collagen. These interactions may target collagen-binding strains of lactobacilli to dentin collagen in the oral cavity and thus play a role in the pathogenesis of root surface and/or coronal caries. Interference with this collagen-mediated attachment of lactobacilli may provide effective means of caries control, particularly in view of the fact that other oral acidogenic microbiota also interact with collagen.

Isolation of glomeruli from areas of bovine cerebellum and comparison of [3H]serotonin uptake.

A method for bulk preparation of glomerular particles from subdivisions of the bovine cerebellum is presented. This method represents a modification of that previously reported by Hajos et al. [8], which increases the yield of glomerular protein by five-fold (5.5 mg/g wet wt) without compromising structural integrity or homogeneity. In addition, it offers the advantage of allowing one to study intraregional variations in the metabolic properties of cerebellar glomeruli. [3H]Serotonin (5-HT) uptake was measured in this preparation and it was demonstrated that glomeruli possess an active high affinity mechanism for this substrate. Comparison of [3H]5-HT uptake by glomeruli isolated from the cerebellar cortices of the lateral hemispheres and vermis revealed no differences in their kinetic properties.

An in vitro model for adhesion of bacteria to human tooth root surfaces.

A model mimicking bacterial colonization of dentine has been developed. It employs uniform particles of pulverized human tooth root tissue incubated with radioactively labelled bacteria. After incubation, the number of attaching bacteria is quantified. Attachment of Streptococcus mutans UA140, Actinomyces viscosus T14, and Lactobacillus casei 101 was found to be time dependent and complete within 1-3 h. Dissociation constants (Kd) of the interactions equalled 2.5 x 10(8) and 1.6 x 10(8) cells/ml, for Strep. mutants and A. viscosus, respectively. The Kd for L. casei could not be determined as attachment was not saturable. The putative tissue components involved in adherence were studied by determining the attachment of bacteria in the presence of competing strains. The results suggest that Strep. mutans and A. viscosus recognized and competed for the same ligand (probably collagen) in the dentine. L. casei attachment did not complete with the attachment of Strep. mutans and A. viscosus. Attachment of all strains was modified by preincubation with saliva and varied with bacterial strain and saliva donor.

A comparison of physiology scores and morphology in a group of patients evaluated for endovascular repair of infrarenal aneurysms.

AIM: Endovascular repair (EVR) of abdominal aortic aneurysms (AAA) is an accepted alternative to open repair (OR). Anatomical suitability for EVR of 196 consecutive AAA referrals is analysed, according to aneurysm size and relative physiological fitness for OR. METHODS: Patients were evaluated for suitability according to 2 sets of arbitrary EVR anatomical criteria: flexible criteria (FC), and a subgroup, rigid criteria (RC) with few technical risks. Suitability was related to aneurysm size and operative risk (POSSUM). RESULTS: Eighty-eight patients (45%) were suitable by FC, 33 (17%) by RC, and 108 (55%) were unsuitable for EVR. Inadequate neck length (56%) and angulation (26%), were principle reasons for unsuitability. Mean AAA diameter was 60.5 mm for the whole group, 58.4 mm for those suitable by FC, 56.5 mm for those suitable by RC and 62.1 mm in those unsuitable (p<0.01). Median physiology scores (interquartile ranges) were 19 (17-21) overall, 18 (17-21) in those suitable FC, 18 (17-19) in those suitable by RC and 19 (18-21) for unsuitable patients (NS). High risk patients with large aneurysms. There were 133 larger aneurysms (=/>55 mm in diameter), of which there were 56 patients with physiology scores =/>20. Of these 16 (29%) and 4 (7%) were suitable by FC and RC, respectively. By comparison, of the remaining 77 with physiology scores of =/<19.35 (45%) were suitable for FC and 15 (19%) for RC (p<0.05). CONCLUSION: Unfit patients with significantly sized aneurysm; ironically those most likely to benefit, tend to be less suitable for EVR.

The experience and training of British general surgeons in trauma surgery for the abdomen, thorax and major vessels.

BACKGROUND: The report Better Carefor the Severely Injured [London: The Royal College of Surgeons of England and the British Orthopaedic Association; 2000] states that an experienced general surgeon trained in the techniques required to perform life-saving emergency surgery is vital in the management of major trauma. The experience and training of general surgeons in the UK in the management of trauma to the abdomen, thorax and major vessels has never been assessed. METHOD: Postal questionnaire sent to UK general surgical consultants and Higher Surgical Trainees (HSTs). RESULTS: A total of 854 (48%) questionnaires were completed. Of respondents, 85% believe that major trauma should be directed to hospitals that provide a dedicated trauma service. Of non-vascular specialists, 43% felt their training was adequate to manage vascular trauma and only one-third of general surgical consultants felt adequately prepared to manage acute cardiothoracic injuries. The median number of trauma laparotomies undertaken annually was 2 for blunt injury and 1 for penetrating injury. Of HSTs, 21% had not performed a splenectomy for trauma and 44% had no experience of packing for liver injuries. CONCLUSIONS: There is limited experience and training in the surgical management of torso trauma in the UK. Implementation of the recommendations from Better Care for the Severely Injured will be hampered unless steps are taken to maximise experience and improve training.

Suprathreshold retinal damage due to single 6 picosecond 1060 nm laser light pulses.

The retinas of rhesus monkeys were exposed to 6 ps laser light pulses at 1060 nm at 62 and 95 muJ. Lesions were examined by light and transmission and scanning electron microscopy. Evidence of damage was seen in all retinal layers, being most severe in the photoreceptor inner and outer segments. In addition to damage mechanisms previously proposed, it is suggested that the peak power flux density achieved with extremely short pulses may lead to some absorbance by inner retinal layers.

Cardiac pathology associated with high sustained +Gz: II. Stress cardiomyopathy.

The myocardial pathology of 14 pigs exposed to HSGz stress of 9 and 15, or 3, 7, and 9 Gz was studied; six control pigs were used as comparisons. Four pigs received propranolol prior to centrifugation and four pigs received atropine. Hearts were studied by light and electron microscopy. Myocardium from stressed pigs showed myofibrillar degeneration, pooling of mitochondria, and cell death. Lesions occurred in random cells of the subendocardium and papillary muscles. Purkinje fibers were also involved. Pretreatment with atropine increased the number of dead cells found and propranolol increased the number of cells showing myofibrillar degeneration. It is postulated that this is a pluricausal cardiomyopathy similar to several experimental conditions. Significance to aerospace medicine is briefly discussed.

Pseudoaneurysm of the internal iliac artery resulting in massive per-rectal bleeding.

Rectal bleeding is a common reason for presentation to hospital, with large bleeds most commonly caused by diverticular disease and angiodysplasia. Here we present an unusual aetiology of massive per-rectal bleeding attributable to pseudoaneurysm of the internal iliac artery leading to an arterial fistula to the distal large bowel. It is hoped the case will serve as a reminder that rectal bleeding can have a less common aetiology.

Conjugation of Y. pestis F1-antigen to gold nanoparticles improves immunogenicity.

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p<0.05) but not in mice immunised with AuNP-F1 in alhydrogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera.

Contact inactivation of orthopoxviruses by household disinfectants.

AIMS: The aim of this study is to identify common household disinfectants that combine significant activity against the type orthopoxvirus, vaccinia virus with minimal impact in terms of potential toxicity and/or damage to household or personal items. METHODS AND RESULTS: Laboratory scale experiments assessed common disinfectants containing anionic and nonionic detergents, oxygen-based bleach, potassium peroxomonosulfate, chloroxylenol or halogenated phenols. Disinfectants were assessed for their ability to inactivate the virus on contact or after a short incubation period in the presence and absence of foetal bovine serum as a potential interferant. Significant differences were observed ranging from negligible effect of detergents to complete inactivation on contact with chloroxylenol. CONCLUSIONS: At least one chloroxylenol-based household disinfectant is available, which inactivates vaccinia virus on contact. SIGNIFICANCE AND IMPACT OF THE STUDY: In the event of a release or major outbreak of a pathogenic orthopoxvirus there is likely to be significant public demand for disinfectants with activity against these viruses. The identification of common household disinfectants with such activity obviates any requirement to stockpile or distribute laboratory/industrial disinfectants for this purpose.

Collagen mediates adhesion of Streptococcus mutans to human dentin.

Some strains of Streptococcus mutans were found to recognize and bind collagen type I. Binding of 125I-labeled collagen type I was specific in that collagen types I and II, but not unrelated proteins, were able to inhibit binding of the labeled ligand to bacteria. Collagen binding to S. mutans was partially reversible and involved a limited number of bacterial binding sites per cell. S. mutans UA 140 cells bound collagen type I with high affinity (Kd = 8 x 10(-8) M). The number of binding sites per cell was 4 x 10(4). Collagen-binding strains of S. mutans were found to adhere to collagen-coated surfaces as well as to pulverized root tissue. S. mutans strains that did not bind the soluble ligand were unable to adhere to these substrata. Adherence to collagen-coated surfaces could be inhibited with collagen or clostridial collagenase-derived collagen peptides. Adherence of S. mutans to dentin was enhanced by collagen types I and II but inhibited by collagen peptides. S. mutans UA 140 bound significantly less 125I-collagen type I following treatment with peptidoglycan-degrading enzymes. These enzymes released a collagen-binding protein (collagen receptor) with a relative molecular size of 16 kDa. The results of this study suggest that collagen mediates adhesion of S. mutans to dentin. This interaction may target collagen-binding strains of S. mutans to dentin in the oral cavity and may play a role in the pathogenesis of root surface caries.

Structural alterations in retinal tissues from rats deficient in vitamin E and selenium and treated with hyperbaric oxygen.

Vitamin E and selenium play key roles in preventing in vitro lipid peroxidation and free radical damage to retinal tissues. In this research, we studied the effects of hyperbaric oxygen on retinal structure in rats fed diets deficient in vitamin E and/or selenium. We also correlated any alterations in retinal structure with previously measured alterations in electroretinograms (ERGs). Age-matched rats were fed a basal diet deficient in both vitamin E and selenium (B diet), a basal diet supplemented with vitamin E alone (B+E diet), or selenium alone (B+Se diet), or with both micronutrients (B+E+Se). Half the rats in each group were treated (+HBO) with hyperbaric oxygen (100% O2 at 3 ATA for 1.5 per hr day, 5 days per week) and half were not (-HBO). We previously found that the rats fed the B diet for 6 weeks and treated with HBO for 4 weeks (B+HBO group) had diminished a-wave ERG amplitudes. At this time point all rats in the B group and half of the rats in the B+E+Se group were killed for the structural studies reported here. Surprisingly, we found no evidence of photoreceptor cell necrosis [i.e. a decreased thickness of the outer nuclear layer (ONL)] in retinas from rats in the B+HBO group despite the diminished amplitude of the a-wave which arises from this retinal layer. Quantitative structural analyses of retinas from rats in the B+HBO, B-HBO, B+E+Se-HBO and B+E+Se+HBO groups also failed to reveal any significant differences in the cell height of the retinal pigmented epithelium (nasal, central or temporal regions) or the number of mitochondria, phagosomas or inclusion bodies in the central retinal pigment epithelium (RPE). The inner nuclear layer (INL) thickness was, however, consistently decreased in all retinal regions for the rats in the B+HBO group. Our previous work also showed that only rats fed the B+Se diet for 17 weeks and treated with HBO for 15 weeks (B+Se+HBO group) showed diminished a-wave and b-wave ERG amplitudes. At this time point rats in the B+E+Se, B+E, and B+Se groups were killed for structural studies reported here. Only rats in the B+Se+HBO group showed a significantly decreased (about 20%) thickness of the central ONL. This evidence of photoreceptor cell necrosis correlated very well with our previous observation of diminished a- and b-wave amplitudes only in the B+Se+HBO group (at week 17).(ABSTRACT TRUNCATED AT 400 WORDS)

Antibiotics alter interactions of Staphylococcus aureus with collagenous substrata.

The effects of sub-minimum inhibitory concentrations (sub-MICs) of three antibiotics affecting the biosynthesis of peptidoglycan on the interactions of Staphylococcus aureus strains with collagenous substrata were evaluated. In a system measuring binding of 125I-labeled collagen, growth of bacteria in the presence of one-quarter MIC of cloxacillin and vancomycin reduced the number of collagen binding sites on the surface of bacteria. Growth in the presence of cefpodoxime reduced the number of collagen binding sites in one strain and increased it in another. Cefpodoxime also increased the dissociation constant of collagen binding to bacteria, 2- to 3-fold, while the other two antibiotics did not affect the affinity of the interaction. In a system measuring adhesion of 125I-labeled bacteria to collagen-coated surfaces or cartilage, bacteria grown in the presence of cloxacillin and vancomycin attached to varying degrees depending on the strain. In contrast, compared to untreated controls as well as to bacteria treated with the other two antibiotics, growth in the presence of cefpodoxime significantly reduced adhesion of the majority of strains tested. Sub-MICs of antibiotics appear to affect staphylococcal adhesion to collagenous substrata with cefpodoxime exhibiting the strongest effect. The critical factor in reducing bacterial adhesion seems not to be the number of bacterial binding sites for collagen, but the affinity of the interaction.

Risk analysis of PCB exposure via the soil-food crop pathway, and alternatives for remediation at Serpukhov, Russian Federation.

A risk assessment was conducted to determine the likelihood of certain health risks resulting from exposure to soils and food crops contaminated with polychlorinated biphenyl (PCBs). PCBs have contaminated soils, river sediments, and air in the past as a result of industrial activities at a capacitor plant located in the City of Serpukhov, Russian Federation. This risk assessment and suggestions for remediation are designed to aid in decision-making efforts by a joint Russian-American research team developing a community, national, and international response to industrial contamination. Bobovnikova et al. (The Science of the Total Environment 139/140, 357-364, [1993]) have reported that PCBs are elevated in soils and sediments, breast milk, and locally grown foods in the Serpukhov area. Data from these and other investigators have been used in this risk assessment to calculate a potential cancer risk resulting from exposure to PCBs. Our assessment indicates that members of the local population may be at increased risk of cancer, and possibly other adverse health effects, as a result of PCB contamination of their environment. Because previously unassessed environmental contamination is a common problem in the former Soviet Republics, as well as many other areas of the world, we believe this type of evaluation, using known methods, can serve as a model for assessment efforts in other parts of the globe and result in remediative efforts in regions constrained by faltering economies.

A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage.

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187-amino-acid repeat motif. Collagen receptor-positive strains adhered to both collagen substrata and cartilage in a time-dependent process. Collagen receptor-specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.

An encephalomyocarditis virus epizootic in a baboon colony.

Approximately 80 baboon deaths were caused by encephalomyocarditis virus (EMCV) infection in a 3060 member research and production colony. The epizootic extended over a 9-month period and occurred in baboons ranging from 1 day to 22 years of age. Acute death was the most common history. When clinical disease was detected, it was characterized by labored respiration associated with acute congestive heart failure. The salient necropsy findings were pulmonary congestion and edema, hydropericardium, hydrothorax, ascites, lymph node and splenic hypertrophy, and pale white-to-tan mottled hearts. The most significant histologic lesion was nonsuppurative necrotizing myocarditis. Placental infection with fetal loss occurred. Diagnosis was confirmed by light microscopy, transmission electron microscopy, virus culture, and serology. Rarely, EMCV-induced antibody persisted in surviving baboons for more than 24 months. EMCV-infected feral rats were the probable source of the virus and their control stopped the epizootic. No EMCV neutralizing antibody was detected in colony support personnel or chimpanzees.