Immune responses to Neisseria gonorrhoeae and implications for vaccine development.

Neisseria gonorrheoae is the causative agent of gonorrhea, a sexually transmitted infection responsible for a major burden of disease with a high global prevalence. Protective immunity to infection is often not observed in humans, possible due to high variability of key antigens, induction of blocking antibodies, or a large number of infections being relatively superficial and not inducing a strong immune response. N. gonorrhoeae is a strictly human pathogen, however, studies using mouse models provide useful insights into the immune response to gonorrhea. In mice, N. gonorrhoea appears to avoid a protective Th1 response by inducing a less protective Th17 response. In mouse models, candidate vaccines which provoke a Th1 response can accelerate the clearance of gonococcus from the mouse female genital tract. Human studies indicate that natural infection often induces a limited immune response, with modest antibody responses, which may correlate with the clinical severity of gonococcal disease. Studies of cytokine responses to gonococcal infection in humans provide conflicting evidence as to whether infection induces an IL-17 response. However, there is evidence for limited induction of protective immunity from a study of female sex workers in Kenya. A controlled human infection model (CHIM) has been used to examine the immune response to gonococcal infection in male volunteers, but has not to date demonstrated protection against re-infection. Correlates of protection for gonorrhea are lacking, which has hampered the progress towards developing a successful vaccine. However, the finding that the Neisseria meningitidis serogroup B vaccines, elicit cross-protection against gonorrhea has invigorated the gonococcal vaccine field. More studies of infection in humans, either natural infection or CHIM studies, are needed to understand better gonococcal protective immunity.

Live attenuated pertussis vaccine for prevention and treatment of allergic airway inflammation in mice.

Live attenuated vaccines often have beneficial non-specific effects, protecting against heterologous infectious and non-infectious diseases. We have developed a live attenuated pertussis vaccine, named BPZE1, currently in advanced clinical development. Here, we examined the prophylactic and therapeutic potential of its pertactin-deficient derivative BPZE1P in a mouse model of house dust mite (HDM)-induced allergic airway inflammation (AAI). BPZE1P was given nasally either before or after sensitization with HDM, followed by HDM challenge, or between two challenge episodes. Vaccination prior to sensitization reduced resistance in the airways, the numbers of infiltrating eosinophils and the concentrations of proinflammatory cytokines, such as IL-1α, IL-1ß and IL-33, in the lungs but had no effect on Th2 cytokine levels. BPZE1P also protected when delivered after sensitization or between two challenge episodes. However, in this case the levels of Th2 cytokines in the lung were decreased without significant effects on IL-1α, IL-1ß and IL-33 production. The vaccine restored lung function and decreased eosinophil influx in the lungs of HDM-treated mice. BPZE1P has a better take than BPZE1 in hosts vaccinated with acellular pertussis vaccines. Therefore, it has interesting potential as a preventive and therapeutic agent against AAI, even in acellular pertussis-vaccinated populations.

Live attenuated Bordetella pertussis vaccine candidate BPZE1 transiently protects against lethal pneumococcal disease in mice.

BPZE1 is a live attenuated vaccine against infection by Bordetella pertussis, the causative agent of whooping cough. It was previously shown that BPZE1 provides heterologous protection in mouse models of disease caused by unrelated pathogens, such as influenza virus and respiratory syncytial virus. Protection was also observed in mouse models of asthma and contact dermatitis. In this study, we demonstrate that BPZE1 also displays protection against an unrelated bacterial pathogen in a mouse model of invasive pneumococcal disease mediated by Streptococcus pneumoniae. While a single administration of BPZE1 provided no protection, two doses of 106 colony-forming units of BPZE1 given in a three-week interval protected against mortality, lung colonization and dissemination in both BALB/c and C57BL/6 mice. Unlike for the previously reported influenza challenge model, protection was short-lived, and waned within days after booster vaccination. Formaldehyde-killed BPZE1 protected only when administered following a live prime, indicating that priming requires live BPZE1 for protection. Protection against mortality was directly linked to substantially decreased bacterial dissemination in the blood and was lost in MyD88 knock-out mice, demonstrating the role of the innate immune system in the mechanism of protection. This is the first report on a heterologous protective effect of the live BPZE1 vaccine candidate against an unrelated bacterial infection.

Pathogenicity and virulence of Bordetella pertussis and its adaptation to its strictly human host.

The highly contagious whooping cough agent Bordetella pertussis has evolved as a human-restricted pathogen from a progenitor which also gave rise to Bordetella parapertussis and Bordetella bronchiseptica. While the latter colonizes a broad range of mammals and is able to survive in the environment, B. pertussis has lost its ability to survive outside its host through massive genome decay. Instead, it has become a highly successful human pathogen by the acquisition of tightly regulated virulence factors and evolutionary adaptation of its metabolism to its particular niche. By the deployment of an arsenal of highly sophisticated virulence factors it overcomes many of the innate immune defenses. It also interferes with vaccine-induced adaptive immunity by various mechanisms. Here, we review data from invitro, human and animal models to illustrate the mechanisms of adaptation to the human respiratory tract and provide evidence of ongoing evolutionary adaptation as a highly successful human pathogen.

Fundamental differences in physiology of Bordetella pertussis dependent on the two-component system Bvg revealed by gene essentiality studies.

The identification of genes essential for a bacterium's growth reveals much about its basic physiology under different conditions. Bordetella pertussis, the causative agent of whooping cough, adopts both virulent and avirulent states through the activity of the two-component system, Bvg. The genes essential for B. pertussis growth in vitro were defined using transposon sequencing, for different Bvg-determined growth states. In addition, comparison of the insertion indices of each gene between Bvg phases identified those genes whose mutation exerted a significantly different fitness cost between phases. As expected, many of the genes identified as essential for growth in other bacteria were also essential for B. pertussis. However, the essentiality of some genes was dependent on Bvg. In particular, a number of key cell wall biosynthesis genes, including the entire mre/mrd locus, were essential for growth of the avirulent (Bvg minus) phase but not the virulent (Bvg plus) phase. In addition, cell wall biosynthesis was identified as a fundamental process that when disrupted produced greater fitness costs for the Bvg minus phase compared to the Bvg plus phase. Bvg minus phase growth was more susceptible than Bvg plus phase growth to the cell wall-disrupting antibiotic ampicillin, demonstrating the increased susceptibility of the Bvg minus phase to disruption of cell wall synthesis. This Bvg-dependent conditional essentiality was not due to Bvg-regulation of expression of cell wall biosynthesis genes; suggesting that this fundamental process differs between the Bvg phases in B. pertussis and is more susceptible to disruption in the Bvg minus phase. The ability of a bacterium to modify its cell wall synthesis is important when considering the action of antibiotics, particularly if developing novel drugs targeting cell wall synthesis.

The evolution of Bordetella pertussis has selected for mutations of acr that lead to sensitivity to hydrophobic molecules and fatty acids.

Whooping cough, or pertussis, is resurgent in numerous countries worldwide. This has renewed interest in Bordetella pertussis biology and vaccinology. The in vitro growth of B. pertussis has been a source of difficulty, both for the study of the organism and the production of pertussis vaccines. It is inhibited by fatty acids and other hydrophobic molecules. The AcrAB efflux system is present in many different bacteria and in combination with an outer membrane factor exports acriflavine and other small hydrophobic molecules from the cell. Here, we identify that the speciation of B. pertussis has selected for an Acr system that is naturally mutated and displays reduced activity compared to B. bronchiseptica, in which the system appears intact. Replacement of the B. pertussis locus with that of B. bronchiseptica conferred higher levels of resistance to growth inhibition by acriflavine and fatty acids. In addition, we identified that the transcription of the locus is repressed by a LysR-type transcriptional regulator. Palmitate de-represses the expression of the acr locus, dependent on the LysR regulator, strongly suggesting that it is a transcriptional repressor that is regulated by palmitate. It is intriguing that the speciation of B. pertussis has selected for a reduction in activity of the Acr efflux system that typically is regarded as protective to bacteria.

A curated genome-scale metabolic model of Bordetella pertussis metabolism.

The Gram-negative bacterium Bordetella pertussis is the causative agent of whooping cough, a serious respiratory infection causing hundreds of thousands of deaths annually worldwide. There are effective vaccines, but their production requires growing large quantities of B. pertussis. Unfortunately, B. pertussis has relatively slow growth in culture, with low biomass yields and variable growth characteristics. B. pertussis also requires a relatively expensive growth medium. We present a new, curated flux balance analysis-based model of B. pertussis metabolism. We enhance the model with an experimentally-determined biomass objective function, and we perform extensive manual curation. We test the model's predictions with a genome-wide screen for essential genes using a transposon-directed insertional sequencing (TraDIS) approach. We test its predictions of growth for different carbon sources in the medium. The model predicts essentiality with an accuracy of 83% and correctly predicts improvements in growth under increased glutamate:fumarate ratios. We provide the model in SBML format, along with gene essentiality predictions.

Bordetella pertussis epidemiology and evolution in the light of pertussis resurgence.

Whooping cough, or pertussis, is resurgent in many countries world-wide. This is linked to switching from the use of whole cell vaccines to acellular vaccines in developed countries. Current evidence suggests that this has resulted in the earlier waning of vaccine-induced immunity, an increase in asymptomatic infection with concomitant increases in transmission and increased selection pressure for Bordetellapertussis variants that are better able to evade vaccine-mediated immunity than older isolates. This review discusses recent findings in B. pertussis epidemiology and evolution in the light of pertussis resurgence, and highlights the important role for genomics-based studies in monitoring B. pertussis adaptation.

Bordetella pertussis evolution in the (functional) genomics era.

The incidence of whooping cough caused by Bordetella pertussis in many developed countries has risen dramatically in recent years. This has been linked to the use of an acellular pertussis vaccine. In addition, it is thought that B. pertussis is adapting under acellular vaccine mediated immune selection pressure, towards vaccine escape. Genomics-based approaches have revolutionized the ability to resolve the fine structure of the global B. pertussis population and its evolution during the era of vaccination. Here, we discuss the current picture of B. pertussis evolution and diversity in the light of the current resurgence, highlight import questions raised by recent studies in this area and discuss the role that functional genomics can play in addressing current knowledge gaps.

Discovery of new populations and DNA barcoding of the Arapahoe snowfly Arsapnia arapahoe (Plecoptera: Capniidae).

The Arapahoe Snowfly, Arsapnia arapahoe (Nelson & Kondratieff)was recently discovered in six different first-order streams outside of the Cache la Poudre River Basin where it was previously considered endemic. Specimens of A. arapahoe were always collected in much lower relative abundance, 1.09% (±2.3SD), than other sympatric adult capniids. The first mitochondrial deoxyribonucleic acid (DNA) barcodes for A. arapahoe and A. coyote (Nelson & Baumann) are presented and compared with those of A. decepta. DNA barcoding was not able to differentiate between A. arapahoe and A. decepta Banks but it was able to indicate that A. coyote is specifically distinct.

Localization of dense intracranial electrode arrays using magnetic resonance imaging.

Intracranial electrode arrays are routinely used in the pre-surgical evaluation of patients with medically refractory epilepsy, and recordings from these electrodes have been increasingly employed in human cognitive neurophysiology due to their high spatial and temporal resolution. For both researchers and clinicians, it is critical to localize electrode positions relative to the subject-specific neuroanatomy. In many centers, a post-implantation MRI is utilized for electrode detection because of its higher sensitivity for surgical complications and the absence of radiation. However, magnetic susceptibility artifacts surrounding each electrode prohibit unambiguous detection of individual electrodes, especially those that are embedded within dense grid arrays. Here, we present an efficient method to accurately localize intracranial electrode arrays based on pre- and post-implantation MR images that incorporates array geometry and the individual's cortical surface. Electrodes are directly visualized relative to the underlying gyral anatomy of the reconstructed cortical surface of individual patients. Validation of this approach shows high spatial accuracy of the localized electrode positions (mean of 0.96 mm ± 0.81 mm for 271 electrodes across 8 patients). Minimal user input, short processing time, and utilization of radiation-free imaging are strong incentives to incorporate quantitatively accurate localization of intracranial electrode arrays with MRI for research and clinical purposes. Co-registration to a standard brain atlas further allows inter-subject comparisons and relation of intracranial EEG findings to the larger body of neuroimaging literature.

Clinical decision support implemented with academic detailing improves prescribing of key renally cleared drugs in the hospital setting.

OBJECTIVE: Lack of dose adjustment for renally cleared drugs in the presence of poor renal function is a common problem in the hospital setting. The absence of a clinical decision support system (CDSS) from direct clinician workflows such as computerized provider order entry (CPOE) hinders the uptake of CDSS. This study implemented CDSS in an environment independent of CPOE, introduced to prescribers via academic detailing, to address the dosing of renally cleared drugs. DESIGN: GFR+ was designed to automatically calculate and update renal function, doses of key drugs adjusted for renal function, and highlight clinically significant decreases in renal function. Prescribers were made aware of GFR+, its navigation, and surrounding clinical issues, using academic detailing. MEASUREMENT: The rate of dosing conformity and management for key renally cleared drugs in hospitalized patients, before and after GFR+ implementation. RESULTS: Improvements were seen in dosing conformity for enoxaparin (from 68% to 86%, p=0.03), gentamicin (63-87%, p=0.01), and vancomycin (47-77%, p=0.07), as well as the appropriate use of gentamicin therapeutic drug monitoring (70-90%, p=0.02). During episodes of acute renal impairment, renally cleared drugs were held on 38% of instances in the pre-intervention period compared with 62% post-intervention (p=0.01). CONCLUSION: Clinical decision support implemented with academic detailing improved dosing conformity and management of key renally cleared drugs in a hospitalized population. Academic detailing should be strongly considered to facilitate the introduction of CDSS systems that cannot be placed directly into the clinician workflow.

Occupant-to-occupant interaction and impact injury risk in side impact crashes.

To date, efforts to improve occupant protection in side impact crashes have concentrated on reducing the injuries to occupants seated on the struck side of the vehicle arising from contact with the intruding side structure and/or external objects. Crash investigations indicate that occupants on the struck side of a vehicle may also be injured by contact with an adjacent occupant in the same seating row. Anecdotal information suggests that the injury consequences of occupant-to-occupant impacts can be severe, and sometimes life threatening. Occupant-to-occupant impacts leave little evidence in the vehicle, and hence these impacts can be difficult for crash investigators to detect and may be underreported. The objective of this study was to evaluate the risk of impact injury from occupant-to-occupant impacts in side impact vehicle crashes. The study examined 9608 crashes extracted from NASS/CDS 1993--2006 to investigate the risk of occupant-to-occupant impacts. The study computed relative risk ratio of serious injury (MAIS 3 or greater) for drivers with and without an adjacent front seat passenger present. This approach avoids uncertainties in the coding of occupant-to-occupant contact. The NASS data showed an 8% increased injury risk for struck side drivers in cases where a belted front seat passenger was present. If the front seat passenger was unbelted, struck side driver injury risk was found to be 30% higher than for struck side drivers without a front seat passenger. A series of 6 full scale vehicle side impact crash tests, both mobile deformable barrier to vehicle and vehicle-to-pole, were conducted to assess injury risk and determine the occupant kinematics which lead to occupant-to-occupant impact. Limitations of the biofidelity of current ATDs to simulate occupant interaction were noted. Occupant interaction indicating risk of serious head injury to both the driver and front seat passenger was observed in vehicle-to-pole side impact. The results show that despite the introduction of countermeasures to protect struck side occupants from contact with intruding structure or external objects, these occupants may be severely injured by impacting adjacent occupants. The feasibility of a potential countermeasure, developed to offer protection for two adjacent occupants as well as a single occupant seated on the non-struck side, was investigated through analysis of the dummy injury responses produced in pole side impact tests, with and without the countermeasure installed. The countermeasure was observed to reduce the risk of head injury from occupant interaction.