Lyssavirus Vaccine with a Chimeric Glycoprotein Protects across Phylogroups.

Rabies is nearly 100% lethal in the absence of treatment, killing an estimated 59,000 people annually. Vaccines and biologics are highly efficacious when administered properly. Sixteen rabies-related viruses (lyssaviruses) are similarly lethal, but some are divergent enough to evade protection from current vaccines and biologics, which are based only on the classical rabies virus (RABV). Here we present the development and characterization of LyssaVax, a vaccine featuring a structurally designed, functional chimeric glycoprotein (G) containing immunologically important domains from both RABV G and the highly divergent Mokola virus (MOKV) G. LyssaVax elicits high titers of antibodies specific to both RABV and MOKV Gs in mice. Immune sera also neutralize a range of wild-type lyssaviruses across the major phylogroups. LyssaVax-immunized mice are protected against challenge with recombinant RABV and MOKV. Altogether, LyssaVax demonstrates the utility of structural modeling in vaccine design and constitutes a broadened lyssavirus vaccine candidate.

Analysis and modeling of coolants and coolers for specimen transportation.

Maintaining cold chain while transporting medical supplies and samples is difficult in remote settings. Failure to maintain temperature requirements can lead to degraded sample quality and inaccuracies in sample analysis. We performed a systematic analysis on different types of transport coolers (polystyrene foam, injection-molded, and rotational molded) and transport coolants (ice, cold packs, frozen water bottles) frequently in use in many countries. Polystyrene foam coolers stayed below our temperature threshold (6°C) longer than almost all other types of coolers, but were not durable. Injection-molded coolers were durable, but warmed to 6°C the quickest. Rotational molded coolers were able to keep temperatures below our threshold for 24 hours longer than injection molded coolers and were highly durable. Coolant systems were evaluated in terms of cost and their ability to maintain cold temperatures. Long lasting commercial cold packs were found to be less cost effective and were below freezing for the majority of the testing period. Frozen plastic water bottles were found to be a reusable and economical choice for coolant and were only below freezing briefly. Finally, we modeled the coolers performance at maintaining internal temperatures below 6°C and built a highly accurate linear model to predict how long a cooler will remain below 6°C. We believe this data may be useful in the planning and design of specimen transportation systems in the field, particularly in remote or resource limited settings.

Lessons from the reestablishment of Public Health Laboratory activities in Puerto Rico after Hurricane Maria.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States' jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies.

Animal and Human Tissue Models of Vertical Listeria monocytogenes Transmission and Implications for Other Pregnancy-Associated Infections.

Intrauterine infections lead to serious complications for mother and fetus, including preterm birth, maternal and fetal death, and neurological sequelae in the surviving offspring. Improving maternal and child heath is a global priority. Yet, the development of strategies to prevent and treat pregnancy-related diseases has lagged behind progress made in other medical fields. One of the challenges is finding tractable model systems that replicate the human maternal-fetal interface. Animal models offer the ability to study pathogenesis and host defenses in vivo However, the anatomy of the maternal-fetal interface is highly divergent across species. While many tools are available to study host responses in the pregnant mouse model, other animals have placentas that are more similar to that of humans. Here we describe new developments in animal and human tissue models to investigate the pathogenesis of listeriosis at the maternal-fetal interface. We highlight gaps in existing knowledge and make recommendations on how they can be filled.

Manikin Human-Patient Simulator Training.

BACKGROUND: Human-patient simulators (HPSs) may help enhance medical education. Manikin HPS devices respond to common field medical interventions, such as cricothyroidotomy, and have realistic feedback features, such as respirations and pulses. This study surveys Special Operations Medics for evaluations of HPS features. METHODS: Of 518 subjects, 376 completed testing and surveys with valid responses. A total of 102 variables were divided into three categories-general characteristics, procedures, and injuries-and assessed on a fivepoint Likert scale. The Student t test was used to analyze data together and as separate groups against each other and against an aggregated mean. RESULTS: Features that received high scores (i.e., higher than 4.5/5) corresponded closely with pillars of the Tactical Combat Casualty Care (TCCC) curriculum, basic life support, and realism. DISCUSSION: US Army Special Operations Command and US Special Operations Command Medics have overall high confidence in manikin HPS devices and specifically in those that align with TCCC training and lifesaving procedures. The skills most valued coincide with difficult-to-practice measures, such as cricothyroidotomy and wound packing. Features such as prerecorded sounds, sex, automated movements, skin color, defibrillation, bowel sounds, and electrocardiogram are rated lower. These evaluations may guide future development or procurement of manikin HPS devices.

InlP, a New Virulence Factor with Strong Placental Tropism.

Intrauterine infection is a major detriment for maternal-child health and occurs despite local mechanisms that protect the maternal-fetal interface from a wide variety of pathogens. The bacterial pathogen Listeria monocytogenes causes spontaneous abortion, stillbirth, and preterm labor in humans and serves as a model for placental pathogenesis. Given the unique immunological environment of the maternal-fetal interface, we hypothesized that virulence determinants with placental tropism are required for infection of this tissue. We performed a genomic screen in pregnant guinea pigs that led to the identification of 201 listerial genes important for infection of the placenta but not maternal liver. Among these genes was lmrg1778 (lmo2470), here named inlP, predicted to encode a secreted protein that belongs to the internalin family. InlP is conserved in virulent L. monocytogenes strains but absent in Listeria species that are nonpathogenic for humans. The intracellular life cycle of L. monocytogenes deficient in inlP (ΔinlP) was not impaired. In guinea pigs and mice, InlP increased the placental bacterial burden by a factor of 3 log10 while having only a minor role in other maternal organs. Furthermore, the ΔinlP strain was attenuated in intracellular growth in primary human placental organ cultures and trophoblasts. InlP is a novel virulence factor for listeriosis with a strong tropism for the placenta. This virulence factor represents a tool for the development of new modalities to prevent and treat infection-related pregnancy complications.

In trans complementation of lethal factor reveal roles in colonization and dissemination in a murine mouse model.

Lethal factor (LF) is a component of the B. anthracis exotoxin and critical for pathogenesis. The roles of LF in early anthrax pathogenesis, such as colonization and dissemination from the initial site of infection, are poorly understood. In mice models of infection, LF-deficient strains either have altered dissemination patterns or do not colonize, precluding analysis of the role of LF in colonization and dissemination from the portal of entry. Previous reports indicate rabbit and guinea pig models infected with LF-deficient strains have decreased virulence, yet the inability to use bioluminescent imaging techniques to track B. anthracis growth and dissemination in these hosts makes analysis of early pathogenesis challenging. In this study, the roles of LF early in infection were analyzed using bioluminescent signature tagged libraries of B. anthracis with varying ratios of LF-producing and LF-deficient clones. Populations where all clones produced LF and populations where only 40% of clones produce LF were equally virulent. The 40% LF-producing clones trans complimented the LF mutants and permitted them to colonize and disseminate. Decreases of the LF producing strains to 10% or 0.3% of the population led to increased host survival and decreased trans complementation of the LF mutants. A library with 10% LF producing clones could replicate and disseminate, but fewer clones disseminated and the mutant clones were less competitive than wild type. The inoculum with 0.3% LF producing clones could not colonize the host. This strongly suggests that between 10% and 0.3% of the population must produce LF in order to colonize. In total, these findings suggest that a threshold of LF must be produced in order for colonization and dissemination to occur in vivo. These observations suggest that LF has a major role in the early stages of colonization and dissemination.

Bacillus anthracis has two independent bottlenecks that are dependent on the portal of entry in an intranasal model of inhalational infection.

Bacillus anthracis can cause inhalational anthrax. Murine inhalational B. anthracis infections have two portals of entry, the nasal mucosa-associated lymphoid tissue (NALT) and the lumen of the lungs. Analysis of the dissemination from these sites is hindered because infections are asynchronous and asymptomatic until the hosts near death. To further understand and compare how B. anthracis disseminates from these two different environments, clonal analysis was employed using a library of equally virulent DNA-tagged clones of a luminescent Sterne strain. Luminescence was used to determine the origin of the infection and monitor the dissemination in vivo. The number of clones and their proportions in the portals of entry, lymph nodes draining the portals, and kidneys were analyzed. Clonal analysis indicated a bottleneck for both portals of entry, yet the extent and location of the reduction in represented clones differed between the routes. In NALT-based infections, all clones were found to germinate in the NALT, but they underwent a bottleneck as the infection spread to the cervical lymph node. However, lung-based infections underwent a bottleneck in a focal region of growth within the lung lumen and did not need to spread through the mediastinal lymph nodes to cause a systemic infection. Further, the average number of clones found in the kidney and the rate at which genetic drift was affecting the disseminated populations were significantly higher in lung-based infections. Collectively, the data suggested that differences in the host environment alter dissemination of B. anthracis depending on the site of initial colonization and growth.

Cellular and physiological effects of anthrax exotoxin and its relevance to disease.

Bacillus anthracis, the causative agent of anthrax, secretes a tri-partite exotoxin that exerts pleiotropic effects on the host. The purification of the exotoxin components, protective antigen, lethal factor, and edema factor allowed the rapid characterization of their physiologic effects on the host. As molecular biology matured, interest focused on the molecular mechanisms and cellular alterations induced by intoxication. Only recently have researchers begun to connect molecular and cellular knowledge back to the broader physiological effects of the exotoxin. This review focuses on the progress that has been made bridging molecular knowledge back to the exotoxin's physiological effects on the host.

Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis.

Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Here, we report that disruption of the gene ftsX, which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms.