An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.

Stability of Chimpanzee Adenovirus Vectored Vaccines (ChAdOx1 and ChAdOx2) in Liquid and Lyophilised Formulations.

Adenovirus vectored vaccines have entered global use during the COVID-19 pandemic, and are in development for multiple other human and veterinary applications. An attraction of the technology is the suitability of the vaccines for storage at 2-8 °C for months. Widely used COVID-19 vaccine ChAdOx1 nCoV-19 (University of Oxford/AstraZeneca) is based on a species E simian adenovirus. Species E simian serotypes have been used in a wide range of other development programs, but the stability of such vectors has not been extensively described in the peer-reviewed literature. Here, we explore the stability of two candidate vaccines based on two species E serotypes: a Rift Valley fever vaccine based upon the ChAdOx1 vector (Y25 serotype) used in ChAdOx1 nCoV-19, and a rabies vaccine based upon a ChAdOx2 vector (AdC68 serotype). We describe each vector's stability in liquid and lyophilised formulations using in vitro and in vivo potency measurements. Our data support the suitability of liquid formulations of these vectors for storage at 2-8 °C for up to 1 year, and potentially for nonrefrigerated storage for a brief period during last-leg distribution (perhaps 1-3 days at 20 °C-the precise definition of acceptable last-leg storage conditions would require further product-specific data). Depending upon the level of inprocess potency loss that is economically acceptable, and the level of instorage loss that is compatible with maintenance of acceptable end-of-storage potency, a previously reported lyophilised formulation may enable longer term storage at 20 °C or storage for a number of days at 30 °C.

Using cross-species vaccination approaches to counter emerging infectious diseases.

Since the initial use of vaccination in the eighteenth century, our understanding of human and animal immunology has greatly advanced and a wide range of vaccine technologies and delivery systems have been developed. The COVID-19 pandemic response leveraged these innovations to enable rapid development of candidate vaccines within weeks of the viral genetic sequence being made available. The development of vaccines to tackle emerging infectious diseases is a priority for the World Health Organization and other global entities. More than 70% of emerging infectious diseases are acquired from animals, with some causing illness and death in both humans and the respective animal host. Yet the study of critical host-pathogen interactions and the underlying immune mechanisms to inform the development of vaccines for their control is traditionally done in medical and veterinary immunology 'silos'. In this Perspective, we highlight a 'One Health vaccinology' approach and discuss some key areas of synergy in human and veterinary vaccinology that could be exploited to accelerate the development of effective vaccines against these shared health threats.

Safety and efficacy of ChAdOx1 RVF vaccine against Rift Valley fever in pregnant sheep and goats.

Rift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus that was first discovered in Kenya in 1930 and has since spread to become endemic in much of Africa and the Arabian Peninsula. Rift Valley fever (RVF) causes recurrent outbreaks of febrile illness associated with high levels of mortality and poor outcomes during pregnancy-including foetal malformations, spontaneous abortion and stillbirths-in livestock, and associated with miscarriage in humans. No vaccines are available for human use and those licensed for veterinary use have potential drawbacks, including residual virulence that may contraindicate their use in pregnancy. To address this gap, we previously developed a simian adenovirus vectored vaccine, ChAdOx1 RVF, that encodes RVFV envelope glycoproteins. ChAdOx1 RVF is fully protective against RVF in non-pregnant livestock and is also under development for human use. Here, we now demonstrate that when administered to pregnant sheep and goats, ChAdOx1 RVF is safe, elicits high titre RVFV neutralizing antibody, and provides protection against viraemia and foetal loss, although this protection is not as robust for the goats. In addition, we provide a description of RVFV challenge in pregnant goats and contrast this to the pathology observed in pregnant sheep. Together, our data further support the ongoing development of ChAdOx1 RVF vaccine for use in livestock and humans.

Testing the Usability of a Software for Geospatial and Transport Modeling in Acute Stroke Service Planning.

Introduction: Geographic visualizations have been used to understand disease since the nineteenth century. We developed a software that creates simple visualizations which can be used as a decision support tool for pre-hospital acute stroke transportation planning. In this study, we test the usability of this software to improve user experience and assess the interpretability of the visualizations it produces as it relates to planning and optimizing stroke systems of care. Materials and Methods: Healthcare practitioners and administrators working within the acute stroke system in Alberta, Canada were invited to participate. Participants were randomized to either the geographic visualization or 2-D temporospatial diagrams. Using a standardized script participants were asked to complete tasks and interpret the visualizations produced by the software. The computer screen and audio were recorded. Recordings were transcribed verbatim and analyzed using inductive thematic analyses. The number of errors made and time to task completion were also analyzed. Results: Eighteen participants (8 physicians, 5 healthcare administrators, 3 paramedics, and 2 nurses) were enrolled. Mean age was 41.22 years (SD: 10.55) and 8 participants were female. It took users a mean of 1.59 min (SD: 0.71) to complete all 10 tasks for the geographic visualizations and a mean of 1.08 min (SD: 0.33) to complete all 15 tasks for the 2-D temporospatial diagrams. Map users made a median of 2 errors (IQR: 4), 2-D temporospatial diagram users also made a median of 2 errors (IQR: 1.5). All but one map user correctly interpreted all maps, only three of the eight 2-D temporospatial diagram correctly interpreted all diagrams. In the qualitative analysis three common themes were identified: comments on the user interface, comments on the visualization tool(s), and suggestions for improvement. Most study participants mentioned that the software would be useful in their work. Conclusions: Healthcare professional from several different aspects of stroke care see geographic visualizations in transport decision making to be a useful tool. The software demonstrated high usability. However, several suggestions were made to improve user experience as well as additional features which could be developed and become the subject of future studies.

Modeling Stroke Patient Transport for All Patients With Suspected Large-Vessel Occlusion.

Importance: Ischemic stroke with large-vessel occlusion can be treated with alteplase and/or endovascular therapy; however, the administration of each treatment is time sensitive. Objective: To identify the optimal triage and transport strategy: direct to the endovascular center (mothership) or immediate alteplase treatment followed by transfer to the endovascular center (drip and ship), for all patients with suspected large-vessel occlusion stroke. Design Setting, and Participants: This was a theoretical, conditional probability modeling study. Existing data from clinical trials of stroke treatment were used for model generation. The study was conducted from February 1, 2017, to March 1, 2018. Main Outcomes and Measures: The time-dependent efficacy of alteplase and endovascular therapy and the accuracy of large-vessel occlusion screening tools were modeled to estimate the probability of positive outcome (modified Rankin Scale score, 0-1 at 90 days) for both the drip-and-ship and mothership transport strategies. Based from onset to treatment, the strategy that estimates the greatest probability of excellent outcome is determined in several different scenarios. Results: The patient's travel time from both thrombolysis and endovascular therapy centers, speed of treatment, and positive predictive value of the screening tool affect whether the drip-and-ship or mothership strategy estimates best outcomes. With optimal treatment times (door-to-needle time: 30 minutes; door-in-door-out time: 50 minutes; door-to-groin-puncture time: 60 minutes [mothership], 30 minutes [drip and ship]), both options estimate similar outcomes when the centers are 60 minutes or less apart. However, with increasing travel time between the 2 centers (90 or 120 minutes), drip and ship is favored if the patient would have to travel past the thrombolysis center to reach the endovascular therapy center or if the patient would arrive outside the alteplase treatment time window in the mothership scenario. Holding other variables constant, if treatment times are slow at the thrombolysis center (door-to-needle time: 60 minutes; door-in-door-out time: 120 minutes), the area where mothership estimates the best outcomes expands, especially when the 2 centers are close together (60 minutes apart or less). The area where mothership estimates the best outcome also expands as the positive predictive value of the screening tool increases. Conclusions and Relevance: This study suggests that decision making for prehospital transport can be modeled using existing clinical trial data and that these models can be dynamically adapted to changing realities. Based on current median treatment times to realize the full benefit of endovascular therapy on a population level, the study findings suggest that delivery of the treatment should be regionally centralized. The study modeling suggests that transport decision making is context specific and the radius of superiority of the transport strategy changes based on treatment times at both centers, transport times, and the triaging tool used.

Eimeria species parasites as novel vaccine delivery vectors: anti-Campylobacter jejuni protective immunity induced by Eimeria tenella-delivered CjaA.

Vaccination of poultry against coccidiosis caused by the Eimeria species is almost entirely based upon varied formulations of live parasites. The recent development of a series of protocols that support genetic complementation by transfection in Eimeria now provides an opportunity to utilise live anticoccidial vaccines to deliver additional vaccinal antigens. The capacity of Eimeria tenella to express an exogenous antigen and induce an immune response during in vivo infection which is protective against subsequent bacterial challenge has been tested here using the anti-Campylobacter jejuni vaccine candidate CjaA. Using restriction enzyme mediated integration (REMI) a transgenic E. tenella population expressing CjaA and the fluorescent reporter mCitrine has been developed. Vaccination of specific pathogen free chickens by single or multiple oral inoculation of E. tenella-CjaA oocysts induced 91% and 86% immune protection against C. jejuni challenge compared with unvaccinated and wild-type E. tenella vaccinated controls (p<0.001). Increasing vaccination number had no significant influence on the magnitude of protection. These results support the hypothesis that eimerian parasites can be developed as multivalent vaccine vectors and encourage the extension of these studies.

Enhanced immune defences in Pacific white shrimp (Litopenaeus vannamei) post-exposure to a vibrio vaccine.

This study was conducted to determine if exposure of shrimp, Litopenaeus vannamei, to a commercial anti-vibrio vaccine caused changes in antibacterial and cellular (phagocytosis) defences. Shrimp post-larvae were administered either Vibromax™ vaccine or a blank preparation. Whole body homogenates were prepared before (day 0), during (day 10) and after (day 20) vaccination and incubated with a selection of pathogenic vibrios. Homogenate from day 0 animals showed natural antibacterial activity towards Vibrioanguillarum which was significantly enhanced for bacteria-exposed shrimp at 10 days post-challenge. This effect of the vaccine was short-term in its duration. No antibacterial activity was observed in day 0 shrimp homogenate against Vibrio alginolyticus but it was significantly enhanced for both vaccinated and blank-vaccinated shrimp by day 10. No natural or inducible antibacterial activity was observed against Vibrio harveyi at 0, 10 or 20 days post-challenge. To determine if prior exposure of shrimp to inactivated vibrios results in elevated hemocyte phagocytic activity, juveniles were injected with either a mixture of formalin-inactivated vibrios or saline. Hemocyte monolayers made from these shrimp were overlaid with a 1:1 mix of Bacillus subtilis and these vibrios. Hemocytes from vibrio-exposed animals showed elevated levels of internalised vibrios compared with those from the saline injected group. These studies show selectively enhanced cellular defences of shrimp following 'vaccination'.

Polymerase chain reaction-based identity assay for pathogenic turkey Eimeria.

Diagnosis of turkey Eimeria infection by conventional parasitologic methods is challenging and, until now, no molecular tools existed that clearly distinguished the four widely recognized pathogenic species: Eimeria adenoeides, E. meleagrimitis, E. gallopavonis, and E. dispersa. In this study, the internal transcribed spacer region one (ITS-1) was amplified and sequenced from 23 conventionally characterized reference samples. Phylogenic analysis segregated samples into five distinct cluster groups. The ITS-1 region(s) within each cluster were of a particular length and shared from 96% to 100% identity, while amplified ITS-1 region(s) between clusters differed in length and shared only 10.6% to 49.7% sequence identity. In addition, we developed PCR primer sets as diagnostic tools capable of specifically identifying members of each of the five clusters.

The strictured anastomosis: successful treatment by corticosteroid injections--report of three cases and review of the literature.

Clinically significant anastomotic strictures usually only occur with very low colorectal anastomoses below the level of the peritoneal reflection. The reported rate averages 8 percent and has been attributed to tissue ischemia, localized sepsis, anastomotic leak, proximal fecal diversion, radiation injury, inflammatory bowel disease, and recurrent rectal cancer. Most patients will have symptoms of obstipation, frequent small bowel movements, and bloating. Symptomatic strictures are often approached by dilation (balloon or Hegar) or less often repeat resection. Many of these patients have anastomoses that are too low to consider repeat resection. Strictureplasty with linear stapling devices, stricture resection by use of the circular stapling device, and repeat dilations have all been described. Steroid injections into the stricture have been described in strictured esophagogastric anastomoses but have not been commonly used for strictured coloproctostomies. We describe three cases of coloanal stricture following resections that were complicated by postoperative pelvic abcesses, anastomatic leaks, and pelvic fibrosis. Two cases had undergone low coloanal anastomosis that was protected by a loop ileostomy and developed as significant stricture in the early postoperative period. The third case was managed without a protective loop ileostomy. These were initially managed by repeated dilation of the anastomosis. Each episode was followed by rapid recurrence of the stricture. All patients underwent subsequent dilation with injection of 40 mg of triamcinolone acetate (divided dose in four quadrants) into the stricture and subsequent complete resolution of the stricture. Those patients with loop ileostomies had them taken down and all have been followed for up to 12 months without clinical or endoscopic evidence of recurrent stricture.

Genomic studies of gene expression: regulation of the Wilson disease gene.

Bacterial artificial chromosomes (BACs) have many advantages over other large-insert cloning vectors and have been used for a variety of genetic applications, including the final contigs of the human genome. We describe the utilization of a BAC construct to study gene regulation in a tissue culture-based system, using a 170-kb clone containing the entire Wilson disease (WND) locus as a model. A second BAC construct that lacked a putative negatively regulating promoter sequence was created. A nonviral method of gene delivery was applied to transfect three human cell lines stably with each construct. Our results show correct WND gene expression from the recombinant locus and quantification revealed significantly increased expression from the clone lacking the negative regulator. Comparison with conventional methods confirms the reliability of the genomic approach for thorough examination of gene expression. This experimental system illustrates the potential of BAC clones in genomic gene expression studies, new gene therapy strategies, and validation of potential molecular targets for drug discovery.

CypHer 5: a generic approach for measuring the activation and trafficking of G protein-coupled receptors in live cells.

GPCRs are one of the most popular classes of therapeutic drug targets. It is therefore important to design specific assay formats to readily identify ligands at these receptors. CypHer 5 technology utilizes the general ability of GPCRs to be internalized into the endosomal pathway of a cell in response to agonist ligands. The CypHer 5 dye is fluorescent in acidic environments, but nonfluorescent at neutral pH. When CypHer 5 is bound to a receptor on the extracellular surface of the cell, it is essentially nonfluorescent. On internalization into a cell, it displays a significant increase in fluorescence. Here we demonstrate the detection of agonist activation of two GPCRs in stably transfected live cells using CypHer 5 technology. The G(q)-coupled TRHR-1 and the G(s)-coupled beta(2)-adrenoceptor were both N-terminally tagged with VSV-G. Following addition of CypHer 5-labeled anti-VSV-G antibodies to HEK 293 cells stably expressing the beta(2)-adrenoceptor or CHO-K1 cells stably expressing the TRHR-1, the cells were treated with agonists and then imaged on Amersham Biosciences' IN Cell Analyzer 3000. Data were quantified using a granularity analysis module. Concentration-response curves were obtained with signal-to-background ratios of 7:1 for both receptors. An EC(50) of 0.52 nM was observed on TRH stimulation of the TRHR-1, and an EC(50) of 30 nM was obtained on isoprenaline stimulation of the beta(2)-adrenoceptor. These results demonstrated that the CypHer technology was capable of measuring high-potency agonist responses. The beta(2)-adrenoceptor antagonist, alprenolol, competed for isoprenaline with an IC(50) of 30 nM, indicating that a high-potency antagonist inhibition curve could also be observed using CypHer. CypHer 5 provides a generic tool to measure GPCR activation in a live cell, homogeneous assay format, and may be equally suitable for detecting activation of other classes of cell surface receptors.

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase.

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.