Improved Efficacy of a Gene Optimised Adenovirus-based Vaccine for Venezuelan Equine Encephalitis Virus

<p>Abstract</p> <p>Background</p> <p>Optimisation of genes has been shown to be beneficial for expression of proteins in a range of applications. Optimisation has increased protein expression levels through improved codon usage of the genes and an increase in levels of messenger RNA. We have applied this to an adenovirus (ad)-based vaccine encoding structural proteins (E3-E2-6K) of Venezuelan equine encephalitis virus (VEEV).</p> <p>Results</p> <p>Following administration of this vaccine to Balb/c mice, an approximately ten-fold increase in antibody response was elicited and increased protective efficacy compared to an ad-based vaccine containing non-optimised genes was observed after challenge.</p> <p>Conclusion</p> <p>This study, in which the utility of optimising genes encoding the structural proteins of VEEV is demonstrated for the first time, informs us that including optimised genes in gene-based vaccines for VEEV is essential to obtain maximum immunogenicity and protective efficacy.</p

Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

<p>Abstract</p> <p>Background</p> <p>There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.</p> <p>Results</p> <p>In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.</p> <p>Conclusion</p> <p>A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.</p> <p>Crown Copyright © 2009</p

The effects of long-term saturated fat enriched diets on the brain lipidome

The brain is highly enriched in lipids, where they influence neurotransmission, synaptic plasticity and inflammation. Non-pathological modulation of the brain lipidome has not been previously reported and few studies have investigated the interplay between plasma lipid homeostasis relative to cerebral lipids. This study explored whether changes in plasma lipids induced by chronic consumption of a well-tolerated diet enriched in saturated fatty acids (SFA) was associated with parallel changes in cerebral lipid homeostasis. Male C57Bl/6 mice were fed regular chow or the SFA diet for six months. Plasma, hippocampus (HPF) and cerebral cortex (CTX) lipids were analysed by LC-ESI-MS/MS. A total of 348 lipid species were determined, comprising 25 lipid classes. The general abundance of HPF and CTX lipids was comparable in SFA fed mice versus controls, despite substantial differences in plasma lipid-class abundance. However, significant differences in 50 specific lipid species were identified as a consequence of SFA treatment, restricted to phosphatidylcholine (PC), phosphatidylethanolamine (PE), alkyl-PC, alkenyl-PC, alkyl-PE, alkenyl-PE, cholesterol ester (CE), diacylglycerol (DG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes. Partial least squares regression of the HPF/CTX lipidome versus plasma lipidome revealed the plasma lipidome could account for a substantial proportion of variation. The findings demonstrate that cerebral abundance of specific lipid species is strongly associated with plasma lipid homeostasis

Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Glucosamine-NISV delivers antibody across the blood-brain barrier : optimization for treatment of encephalitic viruses

The field of brain drug delivery faces many challenges that hinder development and testing of novel therapies for clinically important central nervous system disorders. Chief among them is how to deliver large biologics across the highly restrictive blood-brain barrier. Non-ionic surfactant vesicles (NISV) have long been used as a drug delivery platform for cutaneous applications and have benefits over comparable liposomes in terms of greater stability, lower cost and suitability for large scale production. Here we describe a glucosamine-coated NISV, for blood-brain barrier GLUT1 targeting, capable of traversing the barrier and delivering active antibody to cells within the brain. In vitro, we show glucosamine vesicle transcytosis across the blood-brain barrier with intact cargo, which is partially dynamin-dependent, but is clathrin-independent and does not associate with sorting endosome marker EEA1. Uptake of vesicles into astrocytes follows a more classical pathway involving dynamin, clathrin, sorting endosomes and Golgi trafficking where the cargo is released intracellularly. In vivo, glucosamine-coated vesicles are superior to uncoated or transferrin-coated vesicles for delivering cargo to the mouse brain. Finally, mice infected with Venezuelan equine encephalitis virus (VEEV) were successfully treated with anti-VEEV monoclonal antibody Hu1A3B-7 delivered in glucosamine-coated vesicles and had improved survival and reduced brain tissue virus levels. An additional benefit was that the treatment also reduced viral load in peripheral tissues. The data generated highlights the huge potential of glucosamine-decorated NISV as a drug delivery platform with wider potential applications