The role of nanomaterial-protein interactions in determining the toxic consequences of nanomaterial exposure

As early biological responses to foreign objects in the body can be influenced by their bound proteins, the nanomaterial hard protein corona (the collection of slow exchange proteins that associate with nanomaterials) is an emerging area of interest in nanotoxicology. There is a limited but growing appreciation of the role these interactions have in influencing nanomaterial toxicity. This research dealt with (i) the characterisation of iron oxide and silica particles with and without a plasma, serum and lung lining fluid protein hard corona, (ii) the identification of the proteins in the hard corona that associate with the particles and (iii) the effect of the hard corona on influencing particle cytotoxicity in a J774.A1 macrophage cell line. Initial investigation of the particles illustrates the advantages in using a variety of characterisation techniques to better elucidate particle properties. Subsequent characterisation of the hard corona protein profile demonstrated a clear difference in the biological identity of the particles in a plasma, serum and in a lung lining fluid corona. Although it is difficult to associate the impact of any individual protein identified in the hard corona to cytotoxicity this study indicates that the binding of proteins plays a significant role in altering the cytotoxic potential (as determined by LDH release) in macrophages. The work also demonstrates the hard corona has an impact on macrophage chemotaxis, which further strengthens the hypothesis that the corona is a key consideration in nanoparticle toxicity. Ultimately this thesis finds that the nanomaterial hard corona is an important element to consider in experimental design and highlights the concept of creating particle preparation protocols to mimic the corona composition in vivo when examining in vitro cellular responses. This research highlights the implications for interpretation of data from in vitro cell culture tests that do not take the protein corona into consideration

Nanotoxicology

As the production and use of nanomaterials (NMs) in medicine and many other applications develops, so the need to understand the potential risks posed by NMs to human health (and the environment) increases (Aitken et al. 2006). At the nanoscale (1-100 nm), materials exhibit properties that are different to larger or bulk materials. These new properties are exploited by researchers and industry to generate new products; however, the same properties can also inuence how the NM behaves in biological systems, including affecting toxicity. Nanotoxicology is a relatively new eld of research that aims to assess the human and environmental hazard of nanomaterials. In recent years, this new discipline has seen a rapid expansion in the number of studies concerned with assessing the safety of engineered NMs (Figure 20.1)

The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host

African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV. Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible. We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2. We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection conferred by all other vaccines correlated strongly with the levels of pre-formed AHSV-VP2 in the vaccine inoculum

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene. RESULTS: Culicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site. CONCLUSIONS: Methods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknow

Antiserum from mice vaccinated with modified vaccinia Ankara virus expressing African horse sickness virus (AHSV) VP2 provides protection when it is administered 48h before, or 48h after challenge

AbstractPrevious studies show that a recombinant modified vaccinia Ankara (MVA) virus expressing VP2 of AHSV serotype 4 (MVA-VP2) induced virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR −/−) against challenge. Follow up experiments indicated that passive transfer of antiserum, from MVA-VP2 immune donors to recipient mice 1h before challenge, conferred complete clinical protection and significantly reduced viraemia.These studies have been extended to determine the protective effect of MVA-VP2 vaccine-induced antiserum, when administered 48h before, or 48h after challenge. In addition, passive transfer of splenocytes was undertaken to assess if they confer any degree of immunity to immunologically naïve recipient mice. Thus, antisera and splenocytes were collected from groups of mice that had been vaccinated with MVA-VP2, or wild type MVA (MVA-wt), for passive immunisation of recipient mice. The latter were subsequently challenged with AHSV-4 (together with appropriate vaccinated or unvaccinated control animals) and protection was assessed by comparing clinical signs, lethality and viraemia between treated and control groups. All antiserum recipients showed high protection against disease (100% survival rates even in mice that were immunised 48h after challenge) and statistically significant reduction or viraemia in comparison with the control groups. The mouse group receiving splenocytes from MVA-VP2 vaccinates, showed only a 40% survival rate, with a small reduction in viraemia, compared to those mice that had received splenocytes from MVA-wt vaccinates. These results confirm the primarily humoral nature of protective immunity conferred by MVA-VP2 vaccination and show the potential of administering MVA-VP2 specific antiserum as an emergency treatment for AHSV

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo

Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur

Transplacental Transmission of Bluetongue Virus 8 in Cattle, UK

To determine whether transplacental transmission could explain overwintering of bluetongue virus in the United Kingdom, we studied calves born to dams naturally infected during pregnancy in 2007–08. Approximately 33% were infected transplacentally; some had compromised health. In all infected calves, viral load decreased after birth; no evidence of persistent infection was found

Differential persistence of foot-and-mouth disease virus in African buffalo is related to virus virulence

Foot-and-mouth disease virus (FMDV) circulates as multiple serotypes and strains in many endemic regions. In particular the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbour multiple SAT-serotypes for extended periods in the pharyngeal region. However the exact site and mechanism for persistence remain unclear. FMD in buffaloes offers a unique opportunity to study FMDV-persistence, as transmission from carrier ruminants has only convincingly been demonstrated for this species. Following co-infection of naïve African buffaloes with three SAT-serotypes isolated from field buffaloes; palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days post infection (dpi). Post-mortem examination identified infectious virus for up to 185 dpi and viral genome up to 400 dpi in lymphoid tissue of the head and neck, mainly focussed in germinal centres. Interestingly viral persistence in vivo was not homogenous and the SAT-1 isolate persisted for longer than SAT-2 and SAT-3. Co-infection and passage of these SAT isolates in goat and buffalo cell lines demonstrated a direct correlation between persistence and cell killing capacity. These data suggest FMDV persistence occurs in the germinal centres of lymphoid tissue but the duration of persistence is related to virus replication and cell killing capacity.NJ was funded as a Wellcome Trust Intermediate Clinical Fellow and funding is acknowledged from the Biotechnology and Biological Sciences Research Council (BBS/E/I/00001523 and BBS/E/I/00001717).http://jvi.asm.org2016-11-30hb2016Microbiology and Plant Patholog

Endemic persistence of a highly contagious pathogen : foot-and-mouth disease in its wildlife host

Extremely contagious pathogens are a global biosecurity threat because of their high burden of morbidity and mortality, as well as their capacity for fast-moving epidemics that are difficult to quell. Understanding the mechanisms enabling persistence of highly transmissible pathogens in host populations is thus a central problem in disease ecology. Through a combination of experimental and theoretical approaches, we investigated how highly contagious foot-and-mouth disease viruses persist in the African buffalo, which serves as their wildlife reservoir. We found that viral persistence through transmission among acutely infected hosts alone is unlikely. However, the inclusion of occasional transmission from persistently infected carriers reliably rescues the most infectious viral strain from fade-out. Additional mechanisms such as antigenic shift, loss of immunity, or spillover among host populations may be required for persistence of less transmissible strains.DATA AND MATERIALS AVAILABLITY : Code for our parameter analyses and model simulations are available on Github (https://github.com/SimonGubbins/FMDVInBuffalo/tree/1.0.0 and https://github.com/janmedlock/FMDV/tree/Science_publication) and archived at ZenodoSUPPLEMENTARY MATERIAL 1 : Materials and Methods; Supplementary Text S1 to S6; Figs. S1 to S13; Tables S1 to S7; References (54–83).SUPPLEMENTARY MATERIAL 2 : MDAR Reproducibility Checklist.USDA-NIFA AFRI and by the UK Biotechnology and Biological Sciences Research Council.http://www.sciencemag.orghj2022Microbiology and Plant PathologyVeterinary Tropical Disease