Ferret Interferon (IFN)-Inducible Transmembrane Proteins Are Upregulated by both IFN-α and Influenza Virus Infection.

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, which is 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 genes of humans and mice have 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 interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar 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 and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Evidence for Viral Interference and Cross-reactive Protective Immunity Between Influenza B Virus Lineages.

Background: Two influenza B virus lineages, B/Victoria and B/Yamagata, cocirculate in the human population. While the lineages are serologically distinct, cross-reactive responses to both lineages have been detected. Viral interference describes the situation whereby infection with one virus limits infection and replication of a second virus. We investigated the potential for viral interference between the influenza B virus lineages. Methods: Ferrets were infected and then challenged 3, 10, or 28 days later with pairs of influenza B/Victoria and B/Yamagata viruses. Results: Viral interference occurred at challenge intervals of 3 and 10 days and occasionally at 28 days. At the longer interval, shedding of challenge virus was reduced, and this correlated with cross-reactive interferon γ responses from lymph nodes from virus-infected animals. Viruses from both lineages could prevent or significantly limit subsequent infection with a virus from the other lineage. Coinfections were rare, indicating the potential for reassortment between lineages is limited. Conclusions: These data suggest that innate and cross-reactive immunity mediate viral interference and that this may contribute to the dominance of a specific influenza B virus lineage in any given influenza season. Furthermore, infection with one influenza B virus lineage may be beneficial in protecting against subsequent infection with either influenza B virus lineage.

Phenotype and influx kinetics of leukocytes and inflammatory cytokine production in kidney ischemia/reperfusion injury.

BACKGROUND AND AIM: Kidney ischemia/reperfusion (IR) injury is characterized by tubular epithelial cell (TEC) death and an inflammatory response involving cytokine production and immune cell infiltration. In various kidney diseases, increased macrophage numbers correlate with injury severity and poor prognosis. However, macrophage plasticity enables a diverse range of functions, including wound healing, making them a key target for novel therapies. This study aimed to comprehensively characterize the changes in myeloid and epithelial cells and the production of cytokines throughout the experimental IR model of acute kidney injury to aid in the identification of targets to promote and enhance kidney regeneration and repair. METHODS: Flow cytometric analysis of murine unilateral IR injury was used to assess TEC and myeloid cell subpopulations in conjunction with histological analysis and cytokine production at 6 h, 1, 3, 5 and 7 days post IR injury, spanning the initial inflammatory phase and the following reparative phase. RESULTS: IR injury resulted in a rapid infiltration of Ly6Chigh monocytes and neutrophils with a steady rise in F4/80high MHCIIhigh macrophages over the injury time. The production of the inflammatory cytokines IL-6, MCP-1 and TNF coincided with an increase in IL-10 production. CONCLUSION: This characterization will provide a reference point for future studies designed to manipulate immune cell phenotype and function in order to promote endogenous repair of damaged kidneys.

Allopolyploidy, diversification, and the Miocene grassland expansion.

The role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation.

Establishing the flow cytometric assessment of myeloid cells in kidney ischemia/reperfusion injury.

Polychromatic flow cytometry is a powerful tool for assessing populations of cells in the kidney through times of homeostasis, disease and tissue remodeling. In particular, macrophages have been identified as having central roles in these three settings. However, because of the plasticity of myeloid cells it has been difficult to define a specific immunophenotype for these cells in the kidney. This study developed a gating strategy for identifying and assessing monocyte and macrophage subpopulations, along with neutrophils and epithelial cells in the healthy kidney and following ischemia/reperfusion (IR) injury in mice, using antibodies against CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CSF-1R (CD115), MHC class II, mannose receptor (MR or CD206), an alternatively activated macrophage marker, and the epithelial cell adhesion marker (EpCAM or CD326). Backgating analysis and assessment of autofluorescence was used to extend the knowledge of various cell types and the changes that occur in the kidney at various time-points post-IR injury. In addition, the impact of enzymatic digestion of kidneys on cell surface markers and cell viability was assessed. Comparisons of kidney myeloid populations were also made with those in the spleen. These results provide a useful reference for future analyses of therapies aimed at modulating inflammation and enhancing endogenous remodeling following kidney injury.

Morphological, phylogenetic, and ecological diversity of the new model species Setaria viridis (Poaceae: Paniceae) and its close relatives.

PREMISE OF THE STUDY: Species limits of the emerging model organism Setaria viridis (tribe Paniceae, subtribe Cenchrinae) are not well defined. It is thought to be related to S. adhaerens, S. faberi, S. verticillata, and S. verticilliformis and in North America occurs with the morphologically similar S. pumila. An integrated approach was taken to evaluate its variation and relationships with the other taxa. METHODS: Statistical morphology, flow cytometry, molecular phylogenetics, and growth experiments were employed to examine the group's physical variation, polyploidy, evolutionary relationships, and drought ecology, respectively. KEY RESULTS: SETARIA VIRIDIS contributed one genome to the tetraploids S. faberi, S. verticillata, and S. verticilliformis; the other genome of the latter two was contributed by S. adhaerens. Setaria pumila is unrelated. Morphologically, S. viridis is most similar to S. faberi, but all tested accessions of S. viridis were diploid, whereas those of S. faberi were all tetraploid. Principal component analysis of 70 morphological characters consistently separated S. viridis from S. faberi, largely by spikelet characters. The diagnostic morphological characters are not affected by watering. Setaria faberi is far more sensitive to drought, in terms of mortality and morphological stunting, than S. viridis or S. pumila. CONCLUSIONS: SETARIA VIRIDIS is a diploid species and has contributed to several polyploid derivatives. The most morphologically similar of the polyploids is S. faberi, which differs in spikelet features, phylogenetics, genome size, and ecological response to drought. Researchers using field-collected S. viridis as a model organism will benefit from the clear delimitation provided in this study.

Tolerance strategies for stem-cell-based therapies.

There is much interest in using embryonic stem cells to regenerate tissues and organs. For this approach to succeed, these stem cells or their derivatives must engraft in patients over the long term. Unless a cell transplant is derived from the patient's own cells, however, the cells will be targeted for rejection by the immune system. Although standard methods for suppressing the immune system achieve some success, rejection of the transplant is inevitable. Emerging approaches to address this issue include 're-educating' the immune system to induce tolerance to foreign cells and reducing the immune targeting of the transplant by administering 'self stem cells' instead of foreign cells, but each of these approaches has associated challenges.

Upregulation of RCAN1 causes Down syndrome-like immune dysfunction.

BACKGROUND: People with Down syndrome (DS) are more susceptible to infections and autoimmune disease, but the molecular genetic basis for these immune defects remains undetermined. In this study, we tested whether increased expression of the chromosome 21 gene RCAN1 contributes to immune dysregulation. METHODS: We investigated the immune phenotype of a mouse model that overexpresses RCAN1. RCAN1 transgenic (TG) mice exhibit T cell abnormalities that bear a striking similarity to the abnormalities described in individuals with DS. RESULTS: RCAN1-TG mice display T cell developmental defects in the thymus and peripheral immune tissues. Thymic cellularity is reduced by substantial losses of mature CD4 and CD8 thymocytes and medullary epithelium. In peripheral immune organs T lymphocytes are reduced in number and exhibit reduced proliferative capacity and aberrant cytokine production. These T cell defects are stem cell intrinsic in that transfer of wild type bone marrow into RCAN1-TG recipients restored medullary thymic epithelium and T cell numbers in the thymus, spleen and lymph nodes. However, bone marrow transplantation failed to improve T cell function, suggesting an additional role for RCAN1 in the non-haemopoietic compartment. CONCLUSIONS: RCAN1 therefore facilitates T cell development and function, and when overexpressed, may contribute to immune dysfunction in DS.

A human obesity-associated MC4R mutation with defective Gq/11α signaling leads to hyperphagia in mice.

Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia, and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in WT mice in which a specific Gq/11α inhibitor was delivered to the PVN; this provided evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with limited untoward cardiovascular and other side effects.

The impact of stress and anesthesia on animal models of infectious disease.

Stress and general anesthesia have an impact on the functional response of the organism due to the detrimental effects on cardiovascular, immunological, and metabolic function, which could limit the organism's response to an infectious event. Animal studies have formed an essential step in understanding and mitigating infectious diseases, as the complexities of physiology and immunity cannot yet be replicated in vivo. Using animals in research continues to come under increasing societal scrutiny, and it is therefore crucial that the welfare of animals used in disease research is optimized to meet both societal expectations and improve scientific outcomes. Everyday management and procedures in animal studies are known to cause stress, which can not only cause poorer welfare outcomes, but also introduces variables in disease studies. Whilst general anesthesia is necessary at times to reduce stress and enhance animal welfare in disease research, evidence of physiological and immunological disruption caused by general anesthesia is increasing. To better understand and quantify the effects of stress and anesthesia on disease study and welfare outcomes, utilizing the most appropriate animal monitoring strategies is imperative. This article aims to analyze recent scientific evidence about the impact of stress and anesthesia as uncontrolled variables, as well as reviewing monitoring strategies and technologies in animal models during infectious diseases.

Identification of a novel role for the immunomodulator ILRUN in the development of several T cell subsets in mice.

Inflammation and lipid regulator with UBA-like and NBR1-like domains (ILRUN) is a protein-encoding gene associated with innate immune signaling, lipid metabolism and cancer. In the context of innate immunity, ILRUN inhibits IRF3-mediated transcription of antimicrobial and proinflammatory cytokines by inducing degradation of the transcriptional coactivators CBP and p300. There remains a paucity of information, however, regarding the innate immune roles of ILRUN beyond in vitro analyses. To address this, we utilize a knockout mouse model to investigate the effect of ILRUN on cytokine expression in splenocytes and on the development of immune cell populations in the spleen and thymus. We show elevated production of tumor necrosis factor and interleukin-6 cytokines in ILRUN-deficient splenocytes following stimulation with the innate immune ligands polyinosinic:polycytidylic acid or lipopolysaccharide. Differences were also observed in the populations of several T cell subsets, including regulatory, mucosal-associated invariant and natural killer. These data identify novel functions for ILRUN in the development of certain immune cell populations and support previous in vitro findings that ILRUN negatively regulates the synthesis of pathogen-stimulated cytokines. This establishes the ILRUN knockout mouse model as a valuable resource for further study of the functions of ILRUN in health and disease.

Genomic Characterization of Salmonella enterica serovar Weltevreden Associated with Human Diarrhea.

Salmonella Weltevreden is an emerging pathogen associated with human diarrhea, and knowledge of the genomics and epidemiology of this serovar is still limited. In this study, we performed whole-genome sequencing of 96 S. Weltevreden isolates recovered from diarrheal patients and 62 isolates from food animals in China between 2006 and 2017. Together, with an additional 199 genome sequences of S. Weltevreden published in NCBI, we performed an analysis on all 357 S. Weltevreden genome sequences. Our results demonstrated that the majority of S. Weltevreden from diarrheal patients from China (97.92%, 94/96) and the other regions in the world (94.97%, 189/199) identified in this study were sequence type (ST) 365. The remaining types were ST3771 (n = 3), ST22 (n = 1), ST155 (n = 1), and ST684 (n = 1). In addition, ST365 was also widely recovered from animals, food, and environmental samples in different regions of the world. Phylogenetic analysis and pulsed-field gel electrophoresis (PFGE) revealed that S. Weltevreden from diarrheal patients was closely related to those recovered from food and environmental specimens. We also showed that S. Weltevreden did not exhibit severe antimicrobial resistance profiles, suggesting administering antibiotics is still effective for controlling the agent. Interestingly, we found that S. Weltevreden strains carried a number of virulence factor genes, and a 100.03-kb IncFII(S) type plasmid was widely distributed in S. Weltevreden strains. Elimination of this plasmid decreased the bacterial capacity to infect both Caco-2 cells and C57BL/6 mice, suggesting the importance of this plasmid for bacterial virulence. Our results contribute to the understanding of the epidemiology and virulence of S. Weltevreden. IMPORTANCE 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. In this study, we found S. Weltevreden sequence type (ST) 365 is commonly recovered from diarrheal patients in China and many other regions of the world, and there is no major difference between the Chinese isolates and the global isolates at the phylogenetic level. We also demonstrated that ST365 was widely recovered from animal, food, and environmental samples collected in different, global regions. Importantly, we discovered an IncFII(S) type plasmid commonly carried by S. Weltevreden strains of human, animal, and food origins, and this plasmid is likely to contribute to the bacterial pathogenesis. These findings enhance our understanding of the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.

Biomimetic metal-organic frameworks as protective scaffolds for live-virus encapsulation and vaccine stabilization.

The invaluable health, economic and social impacts of vaccination are hard to exaggerate. The ability to stabilize vaccines is urgently required for their equitable distribution without the dependence on the 'cold-chain' logistics. Herein, for the first time we report biomimetic-mineralization of live-viral vaccines using metal-organic frameworks (MOFs) to enhance their storage stability from days to months. Applying ZIF-8 and aluminium fumarate (Alfum), the Newcastle Disease Virus (NDV) V4 strain and Influenza A WSN strain were encapsulated with remarkable retention of their viral titre. The ZIF-8@NDV, ZIF-8@WSN and Alfum@WSN composites were validated for live-virus recovery using a tissue culture infectious dose (TCID50) assay. With the objective of long-term stabilization, we developed a novel, trehalose (T) and skim milk (SM) stabilized, freeze-dried MOF@Vaccine composite, ZIF-8@NDV+T/SM. The thermal stability of this composite was investigated and compared with the control NDV and non-encapsulated, freeze-dried NDV+T/SM composite at 4 °C, RT, and 37 °C over a period of 12 weeks. We demonstrate the fragility of the control NDV vaccine which lost all viability at RT and 37°C by 12 and 4 weeks, respectively. Comparing the freeze-dried counterparts, the MOF encapsulated ZIF-8@NDV+T/SM demonstrated significant enhancement in stability of the NDV+T/SM composite especially at RT and 37 °C upto 12 weeks. STATEMENT OF SIGNIFICANCE: Vaccination is undoubtedly one of the most effective medical interventions, saving millions of lives each year. However, the requirement of 'cold-chain' logistics is a major impediment to widespread immunization. Live viral vaccines (LVVs) are widely used vaccine types with proven efficacy and low cost. Nonetheless, their complex composition increases their susceptability to thermal stress. Several LVV thermostabilization approaches have been investigated, including their complex engineering and the facile addition of stabilizers. Still, the lack of a universal approach urgently requires finding a stabilization technique especially when additives alone may not be sufficient. Herein, we demonstrate MOF biomimetic-mineralization technology to encapsulate LVVs developing an optimised composite which significantly preserves vaccines without refrigeration for extended periods of time.

Interferon Signaling in Chickens Plays a Crucial Role in Inhibiting Influenza Replication in DF1 Cells.

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.

H7N9 bearing a mutation in the nucleoprotein leads to increased pathology in chickens.

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.

Investigating the Interaction between Negative Strand RNA Viruses and Their Hosts for Enhanced Vaccine Development and Production.

The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host-pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host-pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host-pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.

Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells.

The ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre-clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret-reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

Type I Hypersensitivity in Ferrets Following Exposure to SARS-CoV-2 Inoculum: Lessons Learned.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.