Oncolytic viruses and immune checkpoint inhibitors: the "hot" new power couple

Immune checkpoint inhibitors (ICIs) have revolutionized cancer care and shown remarkable efficacy clinically. This efficacy is, however, limited to subsets of patients with significant infiltration of lymphocytes into the tumour microenvironment. To extend their efficacy to patients who fail to respond or achieve durable responses, it is now becoming evident that complex combinations of immunomodulatory agents may be required to extend efficacy to patients with immunologically “cold” tumours. Oncolytic viruses (OVs) have the capacity to selectively replicate within and kill tumour cells, resulting in the induction of immunogenic cell death and the augmentation of anti-tumour immunity, and have emerged as a promising modality for combination therapy to overcome the limitations seen with ICIs. Pre-clinical and clinical data have demonstrated that OVs can increase immune cell infiltration into the tumour and induce anti-tumour immunity, thus changing a “cold” tumour microenvironment that is commonly associated with poor response to ICIs, to a “hot” microenvironment which can render patients more susceptible to ICIs. Here, we review the major viral vector platforms used in OV clinical trials, their success when used as a monotherapy and when combined with adjuvant ICIs, as well as pre-clinical studies looking at the effectiveness of encoding OVs to deliver ICIs locally to the tumour microenvironment through transgene expression

Antimicrobial use practices and opinions of beef farmers in England and Wales

© British Veterinary Association. Background Limited research exists on antimicrobial use practices of beef farmers. This study aimed to investigate antimicrobial practices and perceptions of beef farmers in England and Wales, and identify drivers of higher antimicrobial use for treatment of bovine pneumonia. Methods A survey was sent out in 2017 to beef farmers in England and Wales who supply to two abattoirs. Descriptive statistics were used to summarise the data. A logistic regression model was built to determine factors associated with treating greater than 5 per cent of the predominant group in the herd with antimicrobials for pneumonia. Results There were a total of 171 useable responses. Most farmers reported using antimicrobials in less than 5 per cent of their herd for the treatment of common diseases. Most farmers (90 per cent) reported that they understood what antimicrobial resistance means, but only 55 per cent were aware of critically important antimicrobials and 9 per cent could name at least one critically important antimicrobial. Having a calf-rearing enterprise and not considering Johne's disease when buying in cattle were associated with using antimicrobials to treat pneumonia in greater than 5 per cent of the predominant group in the herd. Conclusion Self-reported antimicrobial use appears to be low in beef farms. However, some gaps in understanding aspects of antimicrobial stewardship by farmers were identified

Understanding farmers' naturalistic decision making around prophylactic antibiotic use in lambs using a grounded theory and natural language processing approach

The routine use of antibiotics for prevention of disease in neonatal lambs has been highlighted as inappropriate, yet research suggests that many farmers in the UK still carry out this practice.The aim of the study was to understand farmers' naturalistic decision-making around prophylactic antibiotic use in lambs. Data from 431 posts by 133 different users of an online discussion forum were analysed quantitatively using natural language processing and qualitatively using a grounded theory approach. Results from the qualitative analysis identified five categories that influenced farmers risk perceptions around prophylactic antibiotic use in lambs: anticipated regret, negative emotions and experiential avoidance; economic considerations; farmer identity; perception of capability; and perception of social judgement. Natural language processing analysis of the posts by the study group were compared to posts on topics unrelated to antibiotic use by control groups from the same forum to understand the underlying style and tone within the text. Analytical thinking and authenticity scores were significantly lower in the study group compared to the control groups (P [less than] 0.01). Words relating to cognitive processes were significantly higher in the study group compared to the control groups (P [less than 0.01). Results of the qualitative and quantitative analysis were integrated to assess the fit of the data and enhance findings from either method alone.The key findings were the identification of reasons why farmers used antibiotics prophylactically in neonatal lambs. Farmers disassociated the use of oral antibiotics in neonatal lambs from other types of antibiotics within sheep farming. Farmers used the concept of luck and uniqueness to justify their prophylactic antibiotic use. This may explain farmers' low analytical thinking scores as this reflects a more personal style of thinking as farmers express their thoughts on antibiotic use on an individual basis. Farmers' felt that they did not have the capabilities to control neonatal disease without antibiotics during busy periods and this was exacerbated by external economic constraints. For farmers' who used antibiotics for therapeutic use, fear of social judgement was an important factor in their perceptions of risk around antibiotic use. The high frequency of negations and conjunctions suggested feelings of moral duty. As such, antibiotic use has developed into a symbol of their good farming identity which conflicted with the identity held by those who used antibiotics prophylactically. These results can be used to inform knowledge exchange around prophylactic antibiotic use in lambs to improve antibiotic stewardship in the sheep farming sector

To prescribe or not to prescribe? A factorial survey to explore veterinarians’ decision making when prescribing antimicrobials to sheep and beef farmers in the UK

Resistance to antimicrobials is one of the biggest challenges worldwide for public health. A key strategy for tackling this is ensuring judicious use of antimicrobials in human and veterinary medicine. Whilst there are many studies in human medicine investigating prescribing behaviour of doctors, there is limited work to understand what factors influence veterinarian prescribing behaviour. Veterinarians often prescribe antimicrobials to sheep and beef farmers in contexts other than at a clinical consultation, and decision-making behind this has not been explored. The aim of this study was to measure, for the first time, the influence of factors from social theories on veterinarians’ decision to prescribe antimicrobials to sheep and beef farmers without a clinical consultation, using a factorial survey approach. Respondents were presented with eight vignette scenarios, where a farmer asks for antimicrobials at the veterinary practice. Seven factors, identified from constructs of social theories, were included in the vignettes. Random intercept and random slope models were built to estimate the effects of the vignette factors and vet characteristics on the respondents’ willingness to prescribe ratings. A total of 306 surveys were completed. The vignette factors: case type, farmer relationship, other veterinarians in practice, time pressure, habit, willingness to pay, and confidence in the farmer, were significant in the decision to prescribe. Confidence in the farmer was the most influential vignette variable, and was included as a random slope effect. Respondent variables with significant influence on the decision to prescribe were agreeableness personality score, region of veterinary practice, and presence of a small animal department. These influential factors could be considered to target interventions in beef and sheep farm animal veterinary practice for improved antimicrobial stewardship

From the other perspective: Behavioural factors associated with UK sheep farmers’ attitudes towards antibiotic use and antibiotic resistance

Research suggests that many sheep farmers continue to carry out traditional antibiotic use practices despite new’good practice’ recommendations. The aim of this study was to group farmers depending on their attitudes around antibiotic use and antibiotic resistance, and determine the behaviours that are associated with the farmers in these groups. In 2017, a flock health survey was sent to British sheep farmers. K-means cluster analysis was used to identify groups of farmers with similar attitudes towards antibiotic use and resistance. A multivariable logistic regression model was built to determine the associations between farmers’ past behaviours and their antibiotic attitude group. There were 461 responses. Two groups of farmers were identified based on their antibiotic attitudes. Cluster 1 were defined as the "discordant" group who had positive views of using antibiotics prophylactically and negative views of reducing antibiotic use. Cluster 2 were defined as the "concordant" group who were positive about reducing antibiotic use and had negative views about using antibiotics prophylactically. Using antibiotics in all lambs (OR = 2.689, CI = 1.571, 4.603), using antibiotics in all ewes (OR = 3.388, CI = 1.318, 8.706), always trimming diseased feet over the past three years (OR = 2.487, CI = 1.459, 4.238), not using a computer to record information over the past three years (OR = 1.996, CI = 1.179, 3.381), not changing worming practices over the past three years (OR = 1.879, CI = 1.144, 3.087), and farmers’ perceptions that their sheep flock did not make a financial loss in the past three years (OR = 2.088, CI = 1.079, 4.040) were significantly associated with belonging to the discordant group. Talking to their veterinarian about antibiotic use or the frequency of veterinary visits were not associated with antibiotic attitude group. These results suggest that farmers who had attitudes relating to antibiotic use that did not align with current recommendations carried out more traditional practices, which were strengthened by their positive perceptions of profitability

Farmers' Perceptions of Preventing Antibiotic Resistance on Sheep and Beef Farms: Risk, Responsibility, and Action

© 2020 Doidge, Ruston, Lovatt, Hudson, King and Kaler. Antibiotic resistance is one of the most serious public health risks facing humanity. The overuse of antibiotics in the treatment of infectious disease have been identified as sources of the global threat of antibiotic resistance. This paper examines how farmers perceive and manage risks associated with overuse of antibiotics. Specifically, the paper examines the role of habitus and risk in determining farmers' decisions to adopt national antibiotic reduction targets set by members of the Responsible Use of Medicines in Agriculture Alliance's Targets Task Force. Semi-structured interviews were conducted with 34 sheep and beef farmers in England and Wales. Farmers presented four scripts which illuminated reasons for limited adoption of the targets. The scripts presented the farmers as “good farmers” facing an emerging threat to their ontological security. Scripts suggested that they engaged in preventative measures but deflected responsibility for reducing antibiotic resistance to veterinarians and poorly run farms. This research provides valuable insights for policy makers and highlight the benefits of including social science research to support effective implementation

Engineering adenoviral vectors with improved GBM selectivity

Glioblastoma (GBM) is the most common and aggressive adult brain cancer with an average survival rate of around 15 months in patients receiving standard treatment. Oncolytic adenovirus expressing therapeutic transgenes represent a promising alternative treatment for GBM. Of the many human adenoviral serotypes described to date, adenovirus 5 (HAdV-C5) has been the most utilised clinically and experimentally. However, the use of Ad5 as an anti-cancer agent may be hampered by naturally high seroprevalence rates to HAdV-C5 coupled with the infection of healthy cells via native receptors. To explore whether alternative natural adenoviral tropisms are better suited to GBM therapeutics, we pseudotyped an HAdV-C5-based platform using the fibre knob protein from alternative serotypes. We demonstrate that the adenoviral entry receptor coxsackie, adenovirus receptor (CAR) and CD46 are highly expressed by both GBM and healthy brain tissue, whereas Desmoglein 2 (DSG2) is expressed at a low level in GBM. We demonstrate that adenoviral pseudotypes, engaging CAR, CD46 and DSG2, effectively transduce GBM cells. However, the presence of these receptors on non-transformed cells presents the possibility of off-target effects and therapeutic transgene expression in healthy cells. To enhance the specificity of transgene expression to GBM, we assessed the potential for tumour-specific promoters hTERT and survivin to drive reporter gene expression selectively in GBM cell lines. We demonstrate tight GBM-specific transgene expression using these constructs, indicating that the combination of pseudotyping and tumour-specific promoter approaches may enable the development of efficacious therapies better suited to GBM

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells, to suppress MAPK pathway signaling that in turn blocked indirect (gamma interferon release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ regulated gene transcription and lead to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL that resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell specific immune checkpoint, which is responsible for NK cell associated poor outcomes in many cancers

Evaluating the role of CD200 in non-small cell lung carcinoma immune evasion

Lung cancer is the 3rd most common cancer in the UK, accounting for 13% of all new cancer cases and is the most common cause of cancer death in the UK, accounting for 21% of all cancer deaths. Over recent years, the introduction of immunotherapies targeting the PD-1/PDL1 immune checkpoint pathway has revolutionised the treatment of non-small cell lung carcinoma (NSCLC) patients; as a tumour with a typically highly infiltrated immune reactive tumour microenvironment (TME) rich in neoantigens, NSCLC tumours represent an ideal candidate for immunotherapies, as reflected in the durable clinical efficacy seen in some responder patients. However, most patients treated with PD-1/PD-L1 immunotherapies either demonstrate an initial response followed by disease progression or, in the case of 47-63% of patients, demonstrate no response or benefit. Therefore, identification of predictive markers of immune checkpoint inhibitor (ICI) therapy response or the generation of multi-modality immunological therapies targeting multiple immune checkpoints could greatly improve the success of PD-1/PD-L1 ICI therapies in NSCLC. One of the most promising strategies for overcoming resistance and prolonging therapeutic benefit from ICIs is the use of dual immune checkpoint blockade. Therefore, the aim of this thesis was to determine whether the immune checkpoint CD200 may be a relevant immunotherapeutic target in NSCLC patients. CD200 is a type I transmembrane glycoprotein structurally related to the B7 family of receptors that signals through its receptor, CD200R, which is expressed on several immune cell populations including those of myeloid lineage, B cells, NK cells and activated T cells. Engagement of CD200R results in a negative immunoregulatory signal to maintain immune homeostasis and prevent over-activation of the immune system. CD200 is also expressed by several solid and haematological malignancies, with its expression correlating with an increase in immunosuppressive Treg cells, a decrease in memory T cells, and dysfunction of cytotoxic effector NK cells. Furthermore, CD200 is a potent regulator and suppressor of myeloid cell function, suggesting that targeting CD200 signalling may decrease immune suppression and increase the anti-tumour immune response. As a critical regulator of immune homeostasis in normal lung, CD200 may also be expressed by tumour cells to evade immune attack and promote tumour growth. In this thesis, CD200 expression was first characterised in the normal mouse and human lung where it was demonstrated to be strongly expressed throughout the distal lung by both type I and type II alveolar cells. Upon malignant transformation, CD200 expression was demonstrated on both SCC and adenocarcinoma tumours, although to a greater extent in adenocarcinoma tumours. We suggest that type II alveolar cells, the predominant cell of origin v of adenocarcinoma tumours and some SCC tumours, retain CD200 expression upon transformation as a method of immune evasion. Furthermore, using a combined RNA-seq and IHC labelling technique the relationship between tumour CD200 expression and the absolute and relative frequencies of infiltrating immune cells was established. In SCC and adenocarcinoma tumours, CD200 expression positively correlated with the frequencies of immunosuppressive Tregs and in adenocarcinoma tumours, tumour CD200 expression consistently negatively correlated with the frequencies of cytotoxic NK cells. In vitro analysis of CD200R+ NK cell activity demonstrated that CD200+ tumour cells induce NK cell dysfunction as characterised by decreased degranulation, inhibited cytokine production, altered activating receptor expression and a decreased capacity to kill tumour cells. Blocking of CD200 with an antibody was sufficient to restore the NK cell anti-tumour response and increase tumour cell death, suggesting that blocking CD200 signalling in CD200+ tumours could increase NK cellmediated tumour cell killing. Furthermore, single-cell analysis and IHC labelling demonstrated that CD200R is expressed on almost all of the infiltrating immune cell subtypes studied, suggesting immunoregulatory control of the immune response beyond that which was studied in this thesis. Taken together, this data suggests a mechanism by which NSCLC tumour cells express CD200 as a mechanism of immune evasion and that blocking CD200 signalling alone, or in combination with PD-1 inhibition to synergistically increase immune checkpoint therapy efficacy, may represent a novel therapeutic target in NSCLC