Biology subjects

Moffat, K.

Publications and source records attributed to Moffat, K..

11 recordsLinked to original sources

bioRxiv2024

Pathogenesis and immune response to respiratory coronaviruses in their natural porcine host

Porcine respiratory coronavirus (PRCV) is a naturally occurring pneumotropic coronavirus in the pig, providing a valuable large animal model to study acute respiratory disease. PRCV pathogenesis and the resulting immune response was investigated in pigs, the natural large animal host. We compared two strains, ISU-1 and 135, which induced differing levels of pathology in the respiratory tract to elucidate the mechanisms leading to mild or severe disease. The 135 strain induced greater pathology which was associated with higher viral load and stronger spike-specific antibody and T cell responses. In contrast, the ISU-1 strain triggered mild pathology with a more balanced immune response and greater abundance of T regulatory cells. A higher frequency of putative T follicular helper cells was observed in animals infected with strain 135 at 11 days post-infection. Single-cell RNA-sequencing of bronchoalveolar lavage revealed differential gene expression in B and T cells between animals infected with 135 and ISU-1 at 1 day post infection. These genes were associated with cell adhesion, migration, and immune regulation. Along with increased IL-6 and IL-12 production, these data suggest that heightened inflammatory responses to the 135 strain may contribute to pronounced pneumonia. Among BAL immune cell populations, B cells and plasma cells exhibited the most gene expression divergence between pigs infected with different PRCV strains, highlighting their potential role in maintaining immune homeostasis in the respiratory tract. These findings indicate the potential of the PRCV model for studying coronavirus induced respiratory disease and identifying mechanisms that determine infection outcomes. Author summaryUnderstanding how our immune system reacts to respiratory viruses, like SARS-CoV-2, is crucial to developing better treatments. While most COVID-19 infections are mild, some cases lead to severe lung damage, but we do not fully understand why. To study this, we used pigs, which respond more like humans compared to small animals, to explore how the immune system deals with respiratory coronaviruses. We tested two porcine respiratory coronavirus strains that caused different levels of lung damage. The more severe strain triggered a strong immune response and high inflammation, leading to lung pathology similar to that seen in severe COVID-19 cases. By contrast, the milder strain caused a balanced immune response, including more regulatory T cells that help control inflammation. We also found changes in genes related to antibody-producing cells, which may be important for controlling respiratory pathology. Interestingly, changes in immune responses and gene expression lasted long after the virus was cleared, potentially making individuals more vulnerable to future infections - similar to the "long COVID" symptoms seen in people. We propose that this pig model could help us study coronavirus-induced lung damage and test new therapies to prevent severe disease.

immunology↗

bioRxiv2023

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunisation in the pig lung

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leucocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing haemagglutinin and nucleoprotein with or without IL-1{beta}. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1{beta} reduced the number of functionally active Treg. Second, influenza infection upregulated IFI6 in BAL cells, decreasing their susceptibility to virus replication in vitro. Our data provides a reference map of porcine BAL cells and reveals lasting immunological consequences of influenza infection and respiratory immunisation in a highly relevant large animal model for respiratory virus infection. Author SummaryPigs and humans have a similar anatomy and physiology. In humans, cells from lung-washes are used to study immune responses and it was shown that these cells are crucial in protection against respiratory diseases such as influenza and COVID-19. To better understand lung immunity, we compared genes expressed in cells of pig lung-wash to white blood cells, providing an atlas for future studies of immunity in the lung. We also tested a vaccine given to the lung containing IL-1{beta}, a strong immune activator that protects mice against influenza virus infection. However, although IL-1{beta} increased pig immune responses it did not protect pigs against infection. We also showed that the number of immune cells that dampen immune responses (regulatory T cells) is reduced. In addition, we demonstrated increased expression of a protein, IFI6, 21 days after infection showing that while immune cells in the lung have common properties, the invading organisms influence them significantly. Our study elucidates why some vaccines fail despite inducing powerful immune responses, emphasizes the need for caution when applying results from small animals like mice to humans, and indicates the importance of the pig as a model to study disease in humans and livestock.

immunology↗

bioRxiv2023

The mechanical arthropod vector Stomoxys calcitrans influences the outcome of lumpy skin disease virus infection in cattle

The poxvirus lumpy skin disease virus (LSDV) is the etiological agent of lumpy skin disease (LSD), a severe disease of cattle and water buffalo that is characterised by numerous necrotic cutaneous nodules. LSD is a rapidly emerging disease, spreading into and across the Middle East, eastern Europe, and Asia in the past decade. The disease causes substantial production and economic losses in rural communities and affected regions. LSDV is mechanically transmitted by haematophagous arthropods including stable flies (Stomoxys calcitrans), however our understanding of this mechanical transmission method is sparse. A secreted saliva collection methodology using a modified artificial membrane feeding system was optimised for S. calcitrans and used to collect and characterise secreted S. calcitrans saliva. Saliva was mixed with LSDV and shown not to affect virus growth in primary bovine fibroblasts. S. calcitrans saliva or spot-feeding by S. calcitrans was then incorporated into a bovine in vivo experimental model of LSD to determine if either influenced disease pathogenesis. S. calcitrans saliva resulted in fewer animals developing disease, however this difference was not statistically significant. Spot-feeding with S. calcitrans prior to inoculation did not alter the number of animals that developed disease or the overall severity of disease however disease progression was accelerated as demonstrated by the appearance of cutaneous nodules, detection of viral DNA in the blood stream, and production of neutralising antibodies. This shows that S. calcitrans influence disease kinetics through co-incident bite trauma and/or saliva deposition. This increases our understanding of LSDV pathogenesis and highlights the overlooked importance of mechanical vectors in pathogen transmission. Author summaryInsect vectors are important conduits for the transmission of pathogens that cause diseases such as Zika, dengue, malaria, and lumpy skin disease. Biological vector-borne transmission incorporates a replication phase for the pathogen in the insect, whereas no replication occurs in the vector during mechanical transmission. When the insect bites the host it inoculates a pathogen whilst also delivering arthropod-derived factors such as saliva components and causing tissue trauma through biting and probing. Arthropod saliva and/or bite trauma have been shown to enhance the speed and severity of disease following inoculation with a range of biologically transmitted viruses. This study examined if this was true also for the mechanically transmitted pathogen lumpy skin disease virus (LSDV). LSDV is a neglected pathogen that causes severe systemic disease in cattle and is transmitted mechanically by the stable fly Stomoxys calcitrans. Using an experimental bovine model of LSD, we found that disease occurred more rapidly when virus was delivered in association with the bites of uninfected flies. This work has increased our knowledge of lumpy skin disease virus transmission, and the discovery that disease outcome can be impacted by previously overlooked mechanical insect vectors should prompt further investigation into this mechanism of transmission.

microbiology↗

bioRxiv2022

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-{gamma} immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD.

immunology↗

bioRxiv2021

Optimized Multicolour Immunofluorescence Panel for Cattle B Cell Phenotyping by an 8-Colour, 10-Parameter Panel

This 8-colour, 10-parameter panel has been optimised to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterised antibodies against cell surface molecules (immunoglobulin light chain (S-Ig(L)), CD20, CD21, CD40, CD71 and CD138) enabled the discrimination of 24 unique populations within the B cell population. This allows the identification of five putative functionally distinct B cell subsets critical to infection and vaccination responses; 1) naive B cells (BNaive), 2) regulatory B cells (BReg), 3) memory B cells (BMem), 4) plasmablasts (PB) and 5) plasma cells (PC). Although CD3 and CD8 can be included as an additional dump channel, it does not significantly improve the panels ability to separate "Classical" B cells. This panel will promote better characterisation and tracking of B cell responses in cattle as well as other bovid species as the reagents are likely to cross react.

immunology↗

bioRxiv2021

Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence and virulence from attenuated BeninΔDP148R

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, Benin{Delta}DP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from Benin{Delta}DP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (Benin{Delta}DP148R{Delta}EP153R{Delta}EP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from Benin{Delta}DP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with Benin{Delta}DP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.

microbiology↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

immunology↗

bioRxiv2020

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

immunology↗

bioRxiv2019

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The structures reveal a twist of the D-ring, which lead to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signalling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the water dissociation is key to the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

biophysics↗

bioRxiv2017

Comprehensive Benchmarking and Ensemble Approaches for Metagenomic Classifiers

BackgroundOne of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole genome shotgun sequencing data, comprehensive comparisons of these methods are limited. In this study, we use the largest (n=35) to date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of eleven metagenomics classifiers. We also assess the effects of filtering and combining tools to reduce the number of false positives.\n\nResultsTools were characterized on the basis of their ability to (1) identify taxa at the genus, species, and strain levels, (2) quantify relative abundance measures of taxa, and (3) classify individual reads to the species level. Strikingly, the number of species identified by the eleven tools can differ by over three orders of magnitude on the same datasets. However, various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Indeed, leveraging tools with different heuristics is beneficial for improved precision. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species and where customized tools may be required.\n\nConclusionsThe results of this study provide positive controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision and recall. We show that proper experimental design and analysis parameters, including depth of sequencing, choice of classifier or classifiers, database size, and filtering, can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

genomics↗