Daniel S. Layton

The impact of vaccines extends beyond the improvement of individual health, significantly lowering the burden of infectious disease in healthcare systems and strengthening the societal infrastructure and economic stability of a nation. Unfortunately, the substantial benefits of vaccines are often disproportionately encountered in affluent nations, with the need for cold-chain logistics heavily contributing to this inequity. Here, we describe the biomimetic mineralization of a live viral vaccine using metal–organic frameworks (MOFs) to alleviate stress caused by ambient temperatures. Utilizing a zinc-based MOF (ZIF-8), we demonstrated the advantages of a higher concentration of the ZIF-8 precursor constituents for coverage of viral antigens and recovery of the live viral Newcastle Disease Virus (NDV) vaccine. Using a candidate MOF-based formulation, we identified that ZIF-8 can maintain effective coverage of NDV V4 virions following lyophilization; however, viral titer was negatively impacted if rapid freezing was integrated. As an alternative cost-effective storage technique, we utilized filter paper drying for long-term stabilization of various ZIF-8@NDV formulations. We demonstrated that the presence of ZIF-8 significantly contributed to vaccine stability when stored at 25 or 37 °C compared to NDV in solution. In summary, our findings highlight the potential of utilizing MOFs for stabilization of live viral vaccines at ambient temperatures and demonstrate the viability of filter paper as an alternative to lyophilization for vaccine preservation.

Zeolitic imidazolate framework encapsulation of mRNA-loaded lipid nanoparticles significantly enhance transfection efficiency, offering a promising strategy for next-generation mRNA therapeutics.

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology.

Positive-reinforcement training of laboratory pigs can reduce the reliance on forced manual restraint and anaesthesia for sample collection, reducing stress and physiological disruption. Training regimens for laboratory pigs typically rely on specialised equipment for restraint, such as Panepinto slings, with a time investment that may not be justified for short-term studies. These training regimens also commonly rely on pigs being lifted into sling restraints, which is not practical for studies involving large pigs. We developed and assessed a rapid, three-phase, positive-reinforcement training regimen for both individually housed and group-housed laboratory pigs to facilitate the collection of minimally invasive samples consciously and voluntarily. The time to complete each phase of training in both individually housed and group-housed pigs was recorded. The behaviour of the individually housed pigs was assessed via an ethogram of behaviours exhibited during a human approach test, and stress response was assessed by analysing salivary corticosterone. The rapid, positive-reinforcement training regimen successfully facilitated oral swabbing, rectal swabbing and rectal thermometer insertion from individually housed (within 18 days) and group-housed (within 6 days) pigs. The trained pigs displayed increasing positive behaviours, no or very few negative behaviours and corticosterone levels within normal limits throughout the study. This training regimen provides a practical and welfare-positive tool for the collection of minimally invasive samples from both small and large laboratory pigs, with a low time investment of 2–5 min/pig/day without the need for specialised restraint equipment.

Anaesthesia is sometimes required for the effective restraint of laboratory pigs for sample collection. Yet, anaesthesia can initiate a range of physiological disruptions that can increase variability in study data and lead to poorer animal welfare. Judicious use of anaesthesia can mitigate experimental, human safety, and animal welfare concerns, but it does not eliminate the potential for adverse effects. The use of reversal agents can shorten recovery time and reduce the physiological impacts of anaesthesia but can also cause additional side effects. We, therefore, trialled the use of low-dose atipamezole (0.12 mg/kg) for the antagonism of xylazine in laboratory pigs anaesthetised using a combination of xylazine and zolazepam/tiletamine. We measured time to recovery, selected clinical variables, recovery characteristics, and behaviours to investigate if a low dose of antagonist decreased recovery time and reduced the physiological impacts of anaesthesia whilst avoiding adverse negative side effects. We categorised side effects and behaviours as having either a low or high negative welfare impact based on the potential risk of injury and whether behaviours were displayed before or after return to consciousness. Collectively, our results indicated that while the use of low-dose atipamezole decreased recovery time and improved thermoregulation in most pigs, it introduced and exacerbated adverse side effects and behaviours that can lead to poorer welfare outcomes for laboratory pigs.

Salmonella Weltevreden is a pathogen associated with human diarrheal diseases found across the globe. However, knowledge of the genomics and epidemiology of this pathogen is still limited.

The zoonotic H7N9 avian influenza (AI) virus first emerged in 2013 as a low pathogenic (LPAI) strain, and has repeatedly caused human infection resulting in severe respiratory illness and a mortality of ~39% (>600 deaths) across five epidemic waves. This virus has circulated in poultry with little to no discernible clinical signs, making detection and control difficult. Contrary to published data, our group has observed a subset of specific pathogen free chickens infected with the H7N9 virus succumb to disease, showing clinical signs consistent with highly pathogenic AI (HPAI). Viral genome sequencing revealed two key mutations had occurred following infection in the haemagglutinin (HA 226 L>Q) and nucleoprotein (NP 373 A>T) proteins. We further investigated the impact of the NP mutation and demonstrated that only chickens bearing a single nucleotide polymorphism (SNP) in their IFITM1 gene were susceptible to the H7N9 virus. Susceptible chickens demonstrated a distinct loss of CD8⁺ T cells from the periphery as well as a dysregulation of IFNγ that was not observed for resistant chickens, suggesting a role for the NP mutation in altered T cell activation. Alternatively, it is possible that this mutation led to altered polymerase activity, as the mutation occurs in the NP 360-373 loop which has been previously show to be important in RNA binding. These data have broad ramifications for our understanding of the pathobiology of AI in chickens and humans and provide an excellent model for investigating the role of antiviral genes in a natural host species.

Influenza A viruses (IAV) pose a constant threat to human and poultry health. Of particular interest are the infections caused by highly pathogenic avian influenza (HPAI) viruses, such as H5N1, which cause significant production issues. In response to influenza infection, cells activate immune mechanisms that lead to increased interferon (IFN) production. To investigate how alterations in the interferon signaling pathway affect the cellular response to infection in the chicken, we used CRISPR/Cas9 to generate a chicken cell line that lacks a functional the type I interferon receptor (IFNAR1). We then assessed viral infections with the WSN strain of influenza. Cells lacking a functional IFNAR1 receptor showed reduced expression of the interferon stimulated genes (ISG) such as Protein Kinase R (PKR) and Myxovirus resistance (Mx) and were more susceptible to viral infection with WSN. We further investigated the role or IFNAR1 on low pathogenicity avian influenza (LPAI) strains (H7N9) and a HPAI strain (H5N1). Intriguingly, Ifnar^−/− cells appeared more resistant than WT cells when infected with HPAI virus, potentially indicating a different interaction between H5N1 and the IFN signaling pathway. Our findings support that ChIFNAR1 is a key component of the chicken IFN signaling pathway and these data add contributions to the field of host-avian pathogen interaction and innate immunity in chickens.

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM locus of has been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2 and -3 are functionally responsive to both IFN-α and influenza virus stimulation. Thus, we show ferret IFITMs exhibit similar interferon-stimulated properties to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. Importance IFITM proteins can prevent the entry of several pH-dependent viruses, including high consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases, however there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.