Safety and Immunogenicity of Accelerated Heterologous Two-dose Ebola Vaccine Regimens in Adults With and Without HIV in Africa.

BACKGROUND: Shorter prophylactic vaccine schedules may offer more rapid protection against Ebola in resource-limited settings. METHODS: This randomized, observer-blind, placebo-controlled, phase 2 trial conducted in five sub-Saharan African countries included people without HIV (PWOH, n = 249) and people living with HIV (PLWH, n = 250). Adult participants received one of two accelerated Ebola vaccine regimens (MVA-BN-Filo, Ad26.ZEBOV administered 14 days apart [n = 79] or Ad26.ZEBOV, MVA-BN-Filo administered 28 days apart [n = 322]) or saline/placebo (n = 98). The primary endpoints were safety (adverse events [AEs]) and immunogenicity (Ebola virus [EBOV] glycoprotein-specific binding antibody responses). Binding antibody responders were defined as participants with a > 2.5-fold increase from baseline or the lower limit of quantification if negative at baseline. RESULTS: The mean age was 33.4 years, 52% of participants were female, and among PLWH, the median (interquartile range) CD4+ cell count was 560.0 (418.0-752.0) cells/µL. AEs were generally mild/moderate with no vaccine-related serious AEs or remarkable safety profile differences by HIV status. At 21 days post-dose 2, EBOV glycoprotein-specific binding antibody response rates in vaccine recipients were 99% for the 14-day regimen (geometric mean concentrations [GMCs]: 5168 enzyme-linked immunosorbent assay units (EU)/mL in PWOH; 2509 EU/mL in PLWH), and 98% for the 28-day regimen (GMCs: 6037 EU/mL in PWOH; 2939 EU/mL in PLWH). At 12 months post-dose 2, GMCs in PWOH and PLWH were 635 and 514 EU/mL, respectively, for the 14-day regimen and 331 and 360 EU/mL, respectively, for the 28-day regimen. CONCLUSIONS: Accelerated 14- and 28-day Ebola vaccine regimens were safe and immunogenic in PWOH and PLWH in Africa. TRIAL REGISTRATION: NCT02598388.

Strengthening Warfighter Resiliency Using Broad-Spectrum or Host-Directed Therapies within the Rapid Acquisition and Investigation of Drugs for Repurposing Program.

To maintain cadence with looming threats in a prolonged field-care environment, the broader medical countermeasure (MCM) enterprise must adopt new strategies for chemical, biological, radiological, and nuclear (CBRN)-addressing drug development. The Countering Emerging Threats - Rapid Acquisition and Investigation of Drugs for Repurposing (CET RAIDR) program within the Joint Program Manager for CBRN Medical is designed to rapidly tackle known, unknown, and emerging threats by utilizing late-stage or licensed therapeutics. The focus of the CET RAIDR effort is to bridge treatment gaps between threat identification and the implementation of licensed targeted MCMs, thereby strengthening warfighter resiliency. The repurposing approach conserves both time to market and funds by leveraging previous conventional development work as a launch point for repurposing efforts. The CET RAIDR program minimizes development and procurement costs by supplementing the military medical providers' toolbox with post-phase II therapies that demonstrate established safety and manufacturing processes, leading to a cost-sparing model for niche medicines (i.e., CBRN MCMs). SIGNIFICANCE STATEMENT: Countering Emerging Threats - Rapid Acquisition and Investigation of Drugs for Repurposing program candidates are selected based on several pillars: a proven human safety profile, the availability of tools and validated literature on the drug's mechanism of action, well defined assays, and/or animal models to demonstrate efficacy, as well as collaborations with willing and trusted industry partners. This broader repurposing approach to address the growing chemical, biological, radiological, and nuclear threat landscape will better safeguard the warfighter against well documented or unpredictable threats when a direct-acting medical countermeasure is unavailable or not yet conceived.

Serial and parallel organ-at-risk-specific noncoplanar arc optimization for small versus large target volumes in liver SBRT.

Noncoplanar arc optimization has been shown to reduce OAR doses in SRS/SRT and has the potential to reduce doses to OARs in SBRT. Extracranial targets have additional considerations, including large OARs and, in the case of the liver, volume constraints on the healthy liver. Considering pathlengths through OARs that encompass target volumes may lead to specific dose reductions as in the encompassing healthy liver tissue. These optimizations must also leverage delivery efficiency and trajectory sampling to ensure ease of clinical translation. The purpose of this research is to generate optimized static-couch arcs that separately consider serial and parallel OARs and arc delivery efficiency, with a trajectory sampling metric, towards the aim of reducing dose to OARs and the surrounding healthy liver tissue. Separate BEV cost maps were created for parallel, and serial OARs by means of a fast ray-triangle intersection algorithm. An additional BEV cost map was created for the liver which, by definition, encompasses the liver tumors. The individual costs of these maps were summed and combined with the sampling metric for 100 000 random combinations of arc trajectories. A search algorithm was applied to find an arc trajectory solution that satisfied BEV cost and sampling optimization, while also ensuring an efficient delivery was possible with a low number of arcs. This method of arc selection was evaluated for 16 liver SBRT patients characterized by small and large target volumes. Comparisons were made with a clinical arc template of coplanar arcs. Dosimetric plan quality was evaluated using published guidelines and metrics from RTOG1112. Four of five plan quality metrics for the liver were significantly reduced when planned with optimized noncoplanar arcs. Median (range) reductions of the volumes receiving 10, 18, and 21 Gy were found of 140.4 (295.8) cc (p = 0.001), 28.2 (230.6) cc (p = 0.002) and 18.5 (155.5) cc (p = 0.04). A significant increase in median (range) dose to the right kidney of 0.2 ± 0.9 Gy (p = 0.03) was also found using optimized noncoplanar arcs, which was below the tolerance of 10 Gy for all cases. The average number of arcs chosen was 4 ± 1. Optimizing serial and parallel OARs separately during static couch noncoplanar arc selection significantly reduced the dose to the liver during SBRT using a moderate number of arcs.

Carboxyl Methyltransferase Catalysed Formation of Mono- and Dimethyl Esters under Aqueous Conditions: Application in Cascade Biocatalysis.

Carboxyl methyltransferase (CMT) enzymes catalyse the biomethylation of carboxylic acids under aqueous conditions and have potential for use in synthetic enzyme cascades. Herein we report that the enzyme FtpM from Aspergillus fumigatus can methylate a broad range of aromatic mono- and dicarboxylic acids in good to excellent conversions. The enzyme shows high regioselectivity on its natural substrate fumaryl-l-tyrosine, trans, trans-muconic acid and a number of the dicarboxylic acids tested. Dicarboxylic acids are generally better substrates than monocarboxylic acids, although some substituents are able to compensate for the absence of a second acid group. For dicarboxylic acids, the second methylation shows strong pH dependency with an optimum at pH 5.5-6. Potential for application in industrial biotechnology was demonstrated in a cascade for the production of a bioplastics precursor (FDME) from bioderived 5-hydroxymethylfurfural (HMF).

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Safety and Immunogenicity of an Accelerated Ebola Vaccination Schedule in People with and without Human Immunodeficiency Virus: A Randomized Clinical Trial.

The safety and immunogenicity of the two-dose Ebola vaccine regimen MVA-BN-Filo, Ad26.ZEBOV, 14 days apart, was evaluated in people without HIV (PWOH) and living with HIV (PLWH). In this observer-blind, placebo-controlled, phase 2 trial, healthy adults were randomized (4:1) to receive MVA-BN-Filo (dose 1) and Ad26.ZEBOV (dose 2), or two doses of saline/placebo, administered intramuscularly 14 days apart. The primary endpoints were safety (adverse events (AEs)) and immunogenicity (Ebola virus (EBOV) glycoprotein-specific binding antibody responses). Among 75 participants (n = 50 PWOH; n = 25 PLWH), 37% were female, the mean age was 44 years, and 56% were Black/African American. AEs were generally mild/moderate, with no vaccine-related serious AEs. At 21 days post-dose 2, EBOV glycoprotein-specific binding antibody responder rates were 100% among PWOH and 95% among PLWH; geometric mean antibody concentrations were 6286 EU/mL (n = 36) and 2005 EU/mL (n = 19), respectively. A total of 45 neutralizing and other functional antibody responses were frequently observed. Ebola-specific CD4+ and CD8+ T-cell responses were polyfunctional and durable to at least 12 months post-dose 2. The regimen was well tolerated and generated robust, durable immune responses in PWOH and PLWH. Findings support continued evaluation of accelerated vaccine schedules for rapid deployment in populations at immediate risk. Trial registration: NCT02598388 (submitted 14 November 2015).

An evaluation of clinical psychology input into burns multidisciplinary follow-up clinics.

Introduction: Research highlights the complex psychological needs that patients and their families can face following a burn injury, regardless of the objective severity of the injury and often beyond the timeframe of physical healing. Identification of psychological needs at different stages post-burn recovery is therefore a key role of clinical psychologists working in burn care services. Method: This paper presents audit data collected across a two-year period in routine paediatric and adult multidisciplinary team follow-up clinics in a UK burns service. 808 clinical contacts (331 adults, 477 paediatrics) were recorded. Data gathered related to the identification of patient and/or family psychological need and the level of psychology input within clinic. Results: For 43% of adult patients and 46% of paediatric patients seen in clinic, some degree of psychological need for the patient and/or family was identified during the consultation. A large majority of concerns related directly to the burn injury. This is consistent with previous research into the psychological impact of burns. Even for patients with no identified psychological needs, psychology presence enabled the opportunity for brief screening, preventative advice or signposting to take place during clinic. Discussion: A substantial number of individuals and families presented with some level of psychological concern in relation to a burn injury when attending burns multidisciplinary team follow-up clinics. Conclusion: A substantial number of patients and families presented with psychological needs in relation to a burn injury when attending burns MDT follow-up clinics. The presence of Clinical Psychologists at burns MDT follow-up clinics is beneficial for the identification of burns and non-burns related psychological concerns and is a valuable use of psychological resources within a burns service. Lay Summary: The Regional Burns Centre holds regular outpatient scar clinics to monitor recovery and healing. As well as the medical professionals, the clinics are joined by Clinical Psychologists who can assess, refer, and support individuals struggling with their burn or scarring on a mental level. Over 15 months, data was collected about patients attending the clinics and the involvement of the psychologists. 43% of adult patients and 46% of paediatric patients were identified as having some psychological need, either related to their burn or to other aspects of their life. This demonstrates the benefits of having psychology presence within scar clinics, as nearly half of the patients seen in clinic received an assessment and further support (such as signposting and referrals to psychological support). Burns staff also felt that psychology presence enhanced conversations and increased collaboration with decision making around treatment.

Curative Intent Radiation for Anal Cancer in Pelvic Kidney Transplant: A Case Report With an Eight-Year Follow-Up.

The incidence of malignancies seen after solid organ transplant is increasing, and oncologists are seeing more patients with transplanted organs. In this case report, we present how pelvic radiotherapy can be safely administered in a patient with a transplanted kidney by conducting a comprehensive chart review and analyzing the dosimetry in the radiotherapy planning software Eclipse. A 52-year-old female patient received a kidney transplant in 2002 and was diagnosed 11 years later with a cT3 N0 M0 squamous cell carcinoma of the anal canal. She was offered radical radiation therapy with 45 Gy in 25 fractions using a volumetric modulated arc therapy plan to the pelvic lymph nodes and tumor followed by a 9-Gy boost to the anal tumor alone using a three-dimensional conformal radiation therapy plan with concurrent 5-fluorouracil/mitomycin chemotherapy for a total dose of 54 Gy. The right external iliac and inguinal lymph nodes coverage was compromised to decrease the solitary pelvic kidney dose in addition to creating a 1-cm planning risk volume around the kidney and using half-beam blocks. Her pelvic kidney only received a mean dose of 6.68 Gy. Eight years later, the patient continues to be cancer-free, as evident with a recent sigmoidoscopy in 2021 and a physical examination in 2022. Her creatinine started to rise one year post-treatment, but age of the transplanted kidney is likely the cause of kidney failure.

Engineering a Carboxyl Methyltransferase for the Formation of a Furan-Based Bioplastic Precursor.

FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio-derived 2,5-furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5-hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.

Adapting Simon's Two-Stage Design for Efficient Screening of Filovirus Vaccines in Non-Human Primates.

The cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) is widely used for filovirus vaccine testing. To use limited BSL-4 resources efficiently and minimize NHP usage, Simon's two-stage design was adapted to screen candidate Ebola virus (EBOV) vaccines in up to six NHPs with two (optimal), three, or four NHPs in Stage 1. Using the optimal design, two NHPs were tested in Stage 1. If neither survived, the candidate was rejected. Otherwise, it was eligible for Stage 2 testing in four NHPs. Candidates advanced if four or more NHPs were protected over both stages. An 80% efficacious candidate vaccine had 88.5% probability of advancing, and a 40% efficacious candidate vaccine had 83% probability of rejection. Simon's two-stage design was used to screen 27 EBOV vaccine candidates in 43 candidate regimens that varied in dose, adjuvant, formulation, or schedule. Of the 30 candidate regimens tested using two NHPs in Stage 1, 15 were rejected, nine were withdrawn, and six were tested in Stage 2. All six tested in Stage 2 qualified to advance in the product development pipeline. Multiple regimens for the EBOV vaccines approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) in 2019 were tested in this program. This approach may also prove useful for screening Sudan virus (SUDV) and Marburg virus (MARV) vaccine candidates.

A U.S. Government-Coordinated Effort to Leverage Non-Human Primate Data to Facilitate Ebolavirus Vaccine Development.

A United States Government (USG) interagency group, the Filovirus Animal Non-Clinical Group (FANG), has been established to support the development of biodefense medical countermeasures (MCMs). As both vaccines and therapeutics are licensed using "non-traditional pathways", such as the U.S. Food and Drug Administration's (FDA) Animal Rule (AR), non-human primate (NHP) models and associated assays have been developed and standardized across BSL4 testing sites to evaluate candidate products. Vaccine candidates are evaluated using these NHP models, and through this public-private partnership, a meta-analysis of NHP control data has been conducted and submitted to the FDA as a master file. This is an example of how existing NHP control data can be leveraged in lieu of conducting separate natural history studies at multiple testing facilities to demonstrate the consistency of a standardized animal model for vaccine development. As a result, animal use can be minimized and the duplication of effort avoided, thus reducing the amount of time needed to conduct additional studies, as well as the cost of vaccine candidate development. This successful strategy may be applied to other pathogens of high consequence for vaccine development, and shows how strategic preparedness for biodefense can be leveraged in response to outbreaks and public health emergencies.

Determining Planning Priorities for SABR for Oligometastatic Disease: A Secondary Analysis of the SABR-COMET Phase II Randomized Trial.

PURPOSE: SABR may improve survival in patients with oligometastases, but for some lesions, safe delivery of SABR may require a reduction in delivered dose or target coverage. This study assessed the association between target coverage compromise and oncologic and survival outcomes. METHODS AND MATERIALS: Patients with a controlled primary malignancy and 1 to 5 oligometastases were randomized (1:2) between standard of care (SOC) treatment and SOC plus SABR. In patients receiving SABR, the target dose coverage was reduced to meet organ at risk (OAR) constraints, if necessary. The D99 value (minimum dose received by the hottest 99% of the planning target volume [PTV]) was used as a measure of PTV coverage for each treatment plan, and the relationship between the coverage compromise index (CCI, defined as D99/prescription dose) and patient outcomes was assessed. RESULTS: Sixty-two patients in the SABR arm had dosimetric information available and a total of 109 lesions were evaluated. The mean CCI per lesion was 0.96 (95% CI, 0.56-1.61). Of the 109 lesions evaluated, 29.4% (n = 32) required coverage compromise (CCI <0.9). Adrenal metastases required coverage compromise in 100% of analyzed lesions (n = 7). CCI was not significantly associated with lesional control, adverse events, overall survival (OS), or progression-free survival (PFS). CONCLUSIONS: Target compromise was required in a substantial minority of cases, but PTV coverage was not associated with OS, progression-free survival, or lesional control. This suggests that OAR constraints used for SABR treatments in the oligometastatic setting should continue to be prioritized during planning.

Selection of Filovirus Isolates for Vaccine Development Programs.

The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for Zaire ebolavirus. However, effective medical countermeasures, such as vaccines for other filoviruses such as Sudan ebolavirus and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.S. Food and Drug Administration (FDA) Animal Rule. The selection of an appropriate animal model and virus challenge isolates for nonclinical studies are critical aspects of the development program. Here, we have focused on the recommendation of challenge isolates for Sudan ebolavirus and Marburg virus. Based on analyses led by the Filovirus Animal and Nonclinical Group (FANG) and considerations for strain selection under the FDA Guidance for the Animal Rule, we propose prototype virus isolates for use in nonclinical challenge studies.

Natural history of disease in cynomolgus monkeys exposed to Ebola virus Kikwit strain demonstrates the reliability of this non-human primate model for Ebola virus disease.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease.

Toward the combined optimization of dynamic axes (CODA) for stereotactic radiotherapy and radiosurgery using fixed couch trajectories.

PURPOSE: To develop a novel system for patient-specific combined optimization of couch, collimator, and gantry angles for use in volumetric modulated arc therapy (VMAT) treatment planning. The system was designed to produce highly compact dose distributions by extensively sampling the 4π space. Automated fixed couch trajectory planning was used to reduce normal tissue doses by avoiding beams-eye-view (BEV) overlap with organs-at-risk (OARs) and improve monitor unit (MU) efficiency through collimator angle optimization. METHODS: By merging distinct BEV objective functions used to optimize the couch rotation angle and collimator angle, a three-dimensional (3D) cost space (the CODA cube) was constructed with axes of gantry, couch, and collimator rotation angles. At each voxel in this CODA cube, the cost of implementing this combination of axes positions in fixed couch trajectories was quantified. The CODA cube was sampled and explored using a modified constrained Bellman-Ford algorithm to suggest low-cost fixed candidate arcs on each plane of the space, from which 10-arcs are chosen throughout the 3D space using a k-means clustering algorithm. These fixed couch trajectories were then imported into the Eclipse treatment planning system (v.11) and inverse-optimized according to clinical standards. Eight artificial cranial targets were contoured in a test-patient anatomy, and seven treatment plans were generated from combinations of three and four targets. The CODA cube optimized plans were compared to standard 4-arc VMAT plans for cranial stereotactic radiotherapy/surgery that were optimized for the same sets of targets; maximum dose to each OAR, V12Gy to normal brain, conformity, and total MUs were compared. Both planning methods were inverse-optimized with identical dosimetric objectives. RESULTS: CODA plans resulted in a reduction in maximum dose to OARs of 20.6% (P < 0.01), with maximum brainstem dose decreased by 2.63 Gy (P = 0.031) on average when compared to the standard arc arrangement. The mean reduction in total MU was 8.6% (P = 0.156), the mean increase in the inverse of the van't Riet conformation number was 0.1%, (P = 0.67) and the mean decrease in normal brain tissue receiving 12 Gy or higher was 3.9% (P = 0.16), when compared to the standard VMAT arc configuration (n = 7). CONCLUSIONS: The optimization of couch, collimator, and gantry angles simultaneously using a 3D optimization space achieved improvement on multiple clinical metrics when compared to the standard VMAT arc configuration. A statistically significant sparing to OAR maximum doses was seen. Combining these optimizations may yield superior results to independent optimization.

Safety and immunogenicity of a highly attenuated rVSVN4CT1-EBOVGP1 Ebola virus vaccine: a randomised, double-blind, placebo-controlled, phase 1 clinical trial.

BACKGROUND: The safety and immunogenicity of a highly attenuated recombinant vesicular stomatitis virus (rVSV) expressing HIV-1 gag (rVSVN4CT1-HIV-1gag1) was shown in previous phase 1 clinical studies. An rVSV vector expressing Ebola virus glycoprotein (EBOV-GP) in place of HIV-1 gag (rVSVN4CT1-EBOVGP1) showed single-dose protection from lethal challenge with low passage Ebola virus in non-human primates. We aimed to evaluate the safety and immunogenicity of the rVSVN4CT1-EBOVGP1 vaccine in healthy adults. METHODS: We did a randomised double-blind, placebo-controlled, phase 1 dose-escalation study at a single clinical site (Optimal Research) in Melbourne, FL, USA. Eligible participants were healthy men and non-pregnant women aged 18-60 years, with a body-mass index (BMI) of less than 40 kg/m2, no history of filovirus infection, VSV infection, or receipt of rVSV in previous studies, and who had not visited regions where Ebola virus outbreaks have occurred. Three cohorts were enrolled to assess a low (2·5 × 104 plaque forming units [PFU]), intermediate (2 × 105 PFU), or high dose (1·8 × 106 PFU) of the vaccine. Participants within each cohort were randomly allocated (10:3) to receive vaccine or placebo by intramuscular injection in a homologous prime and boost regimen, with 4 weeks between doses. All syringes were masked with syringe sleeves; participants and study site staff were not blinded to dose level but were blinded to active vaccine and placebo. The primary outcomes were safety and tolerability; immunogenicity, assessed as GP-specific humoral immune response (at 2 weeks after each dose) and cellular immune response (at 1 and 2 weeks after each dose), was a secondary outcome. All randomised participants were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, NCT02718469. FINDINGS: Between Dec 22, 2015, and Sept 15, 2016, 39 individuals (18 [46%] men and 21 [54%] women, mean age 51 years [SD 10]) were enrolled, with ten participants receiving the vaccine and three participants receiving placebo in each of three cohorts. One participant in the intermediate dose cohort was withdrawn from the study because of a diagnosis of invasive ductal breast carcinoma 24 days after the first vaccination, which was considered unrelated to the vaccine. No severe adverse events were observed. Solicited local adverse events occurred in ten (26%) of 39 participants after the first dose and nine (24%) of 38 participants after the second dose; the events lasted 3 days or less, were predominantly injection site tenderness (17 events) and injection site pain (ten events), and were either mild (19 events) or moderate (ten events) in intensity. Systemic adverse events occurred in 13 (33%) of 39 participants after the first dose and eight (21%) of 38 participants after the second dose; the events were mild (45 events) or moderate (11 events) in severity, and the most common events were malaise or fatigue (13 events) and headache (12 events). Arthritis and maculopapular, vesicular, or purpuric rash distal to the vaccination site(s) were not reported. A GP-specific IgG response was detected in all vaccine recipients after two doses (and IgG response frequency was 100% after a single high dose), and an Ebola virus neutralising response was detected in 100% of participants in the high-dose cohort. INTERPRETATION: The rVSVN4CT1-EBOVGP1 vaccine was well tolerated at all dose levels tested and was immunogenic despite a high degree of attenuation. The combined safety and immunogenicity profile of the rVSVN4CT1-EBOVGP1 vaccine vector support phase 1-2 clinical evaluation. FUNDING: US Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense: Joint Project Manager for Chemical, Biological, Radiological and Nuclear Medical.

Effect of tillage and rainfall on transport of manure-applied Cryptosporidium parvum oocysts through soil.

Most waterborne outbreaks of cryptosporidiosis have been attributed to agricultural sources due to the high prevalence of Cryptosporidium oocysts in animal wastes and manure spreading on farmlands. No-till, an effective conservation practice, often results in soil having higher water infiltration and percolation rates than conventional tillage. We treated six undisturbed no-till and six tilled soil blocks (30 by 30 by 30 cm) with 1 L liquid dairy manure containing 10(5) C. parvum oocysts per milliliter to test the effect of tillage and rainfall on oocyst transport. The blocks were subjected to rainfall treatments consisting of 5 mm or 30 mm in 30 min. Leachate was collected from the base of the blocks in 35-mL increments using a 64-cell grid lysimeter. Even before any rain was applied, approximately 300 mL of water from the liquid manure (30% of that applied) was transported through the no-till soil, but none through the tilled blocks. After rain was applied, a greater number and percentage of first leachate samples from the no-till soil blocks compared to the tilled blocks tested positive for Cryptosporidium oocysts. In contrast to leachate, greater numbers of oocysts were recovered from the tilled soil, itself, than from the no-till soil. Although tillage was the most important factor affecting oocyst transport, rainfall timing and intensity were also important. To minimize transport of Cryptosporidium in no-till fields, manure should be applied at least 48 h before heavy rainfall is anticipated or methods of disrupting the direct linkage of surface soil to drains, via macropores, need to be used.

Development of a highly sensitive in vitro endothelial cell toxicity assay for evaluating ricin toxin A chain-based vaccines or therapeutics.

The ricin toxin A chain (RTA) is responsible for ricin intoxication due to inhibition of protein synthesis. RTA is also known to cause endothelial toxicity [via a 3 amino acid sequence (x)D(y) motif that acts as a natural disintegrin] resulting in vascular leak syndrome (VLS) in humans. An in vitro endothelial cell toxicity (ECT) assay was developed to evaluate if the ricin vaccine candidate (RVEc) exhibited endothelial toxicity, determined by altered transendothelial electrical resistance (TEER) across human umbilical vein endothelial cell (HUVEC) monolayers. Timepoints at 2 and 4 h were included to evaluate HUVEC monolayers before the effects of RTA ribotoxic activity are observed. Both the 3 µM and 6 µM RTA positive controls consistently demonstrated significantly reduced TEER values, compared to their corresponding vehicle control, in a time- and concentration-dependent manner at 2, 4, and 24 h. Fluorescent imaging of HUVECs exposed to 3 µM RTA showed cell rounding at 2 and 4 h and gap formation at 24 h. No changes in TEER or fluorescent imaging were observed after exposure to endothelial cell growth medium-2 (EGM-2) exchange (mock control). The negative controls, which included 2 mutant RTA vaccine derivatives [RVEc with an (x)D(y) VLS sequence modification to V76M or D75N] and bovine serum albumin (BSA), demonstrated no evidence of HUVEC toxicity at 3 µM and 6  µM concentrations. Overall, the performance of the ECT assay was consistent, allowing for the development of acceptance criteria that were related to time- and concentration-dependent decreases in TEER between 2 and 24 h.

Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule.

Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the "Alphavirus Workshop" organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.

Intra-arc binary collimation algorithm for the optimization of stereotactic radiotherapy treatment of multiple metastases with multiple prescriptions.

PURPOSE: To design and implement a novel treatment planning algorithm based on a modification of dynamic conformal arc (DCA) therapy for the treatment of multiple cranial metastases with variable prescription doses. METHODS: A workflow was developed in which separate dose matrices were calculated for each target at each control point (i.e., the multileaf collimator (MLC) was fit conformally to that single target). A cost function was used to quantify the relative contributions of each dose matrix in the plan to the overall plan objectives. Simulated annealing was used to allow for the inclusion or exclusion of individual dose matrices at each control point. The exclusion of individual targets at a given control point is termed intra-arc binary collimation (iABC) in this work and is accomplished by closing the MLCs over the target for a duration specified by simulated annealing optimization. Dynamic collimator motions were employed to minimize the variation between the idealized dose matrices (i.e., perfectly collimated targets) and actual dose matrices (i.e., MLC apertures that include quantities of nontarget tissue due to the relative orientations of targets in the field). An additional simulated annealing optimization was performed to weight the relative contributions of dose at each control point [referred to as the monitor unit distribution (MUD)] to improve compliance with plan objectives. The algorithm was tested on seven previously treated multiple metastases patients and plans were compared to the clinically treated VMAT plans. RESULTS: Treatment plans generated with iABC used an average of 2716 (34%) fewer MU in the total plan than VMAT (P = 0.01). All normal tissue metrics for all plans and all patients were clinically acceptable. There were no statistically significant differences in any normal tissue dose metrics. Normalized prescription target coverage accuracy for all targets was 3% better on average for VMAT plans when compared to iABC (P = 0.07), and 14% better on average for iABC when compared to optimized DCA (P = 0.03). CONCLUSION: A novel method of aperture and dose distribution design has been developed to significantly increase the MU efficiency of single isocenter treatment of multiple metastases with variable prescription doses when compared to VMAT, and which improves target coverage accuracy significantly when compared to optimized DCA. By applying a DCA approach to subsets of targets across control points, a hybrid method of treatment delivery has been developed that combines the efficiency of dynamic conformal treatments and the dosimetric flexibility of VMAT.