Infection with Seasonal H1N1 Influenza Results in Comparable Disease Kinetics and Host Immune Responses in Ferrets and Golden Syrian Hamsters.

Animal models of influenza are important in preclinical research for the study of influenza infection and the assessment of vaccines, drugs and therapeutics. Here, we show that Golden Syrian hamsters (Mesocricetus auratus) inoculated via the intranasal route with high dose of influenza H1N1 display comparable disease kinetics and immune responses to the 'gold standard' ferret (Mustela furo) model. We demonstrate that both the hamster and ferret models have measurable disease endpoints of weight loss, temperature change, viral shedding from the upper respiratory tract and increased lung pathology. We also characterised both the humoral and cellular immune responses to infection in both models. The comparability of these data supports the Golden Syrian hamster model being useful in preclinical evaluation studies to explore the efficacy of countermeasures against influenza.

Sequential Delivery of Live Attenuated Influenza Vaccine and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the Ferret Model Can Reduce SARS-CoV-2 Shedding and Does Not Result in Enhanced Lung Pathology.

Cocirculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in coinfected patients. The initial lack of a readily available coronavirus disease 2019 (COVID-19) vaccine has reinforced the importance of influenza vaccine programs during the COVID-19 pandemic. Live attenuated influenza vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration influences the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV was administered to ferrets 3 days before or after SARS-CoV-2 infection. LAIV administration did not exacerbate the SARS-CoV-2 disease course or lung pathology with either regimen. In addition, LAIV administered before SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract. This study demonstrated that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

Severity of heterosubtypic influenza virus infection in ferrets is reduced by live attenuated influenza vaccine.

Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

Heterosubtypic cross-protection correlates with cross-reactive interferon-gamma-secreting lymphocytes in the ferret model of influenza.

An effective universal vaccine for influenza will likely need to induce virus-specific T-cells, which are the major mediator of heterosubtypic cross-protection between different subtypes of influenza A virus. In this study we characterise the cell-mediated immune response in ferrets during heterosubtypic protection induced by low-dose H1N1 virus infection against an H3N2 virus challenge, given 4 weeks later. Although the ferrets were not protected against the infection by H3N2 virus, the duration of virus shedding was shortened, and clinical disease was markedly reduced. No cross-reactive neutralizing antibodies were detected, but cross-reactive interferon-gamma-secreting T cells were detected in the circulation prior to H3N2 challenge. These T-cells peaked at 11 days post-H1N1 infection, and were strongly induced in blood and in lung following H3N2 infection. The rapid induction of interferon-gamma-secreting cells in ferrets previously infected with H1N1 virus, but not in naïve ferrets, suggests induction of memory T-cells. These results are in accord with the observations that pre-existing cross-reactive T-cells correlate with protection in humans and have implications for outbreak modelling and universal vaccine design.

Cellular immune response to human influenza viruses differs between H1N1 and H3N2 subtypes in the ferret lung.

Seasonal influenza virus infections cause yearly epidemics which are the source of a significant public health burden worldwide. The ferret model for human influenza A virus (IAV) is widely used and has several advantages over other animal models such as comparable symptomology, similar receptor distribution in the respiratory tract to humans and the ability to be infected with human isolates without the need for adaptation. However, a major disadvantage of the model has been a paucity of reagents for the evaluation of the cellular immune response. Investigation of T-cell mediated immunity in ferrets is crucial to vaccine development and efficacy studies. In this study we have used commercially produced antibodies to ferret interferon gamma (IFN-γ) allowing us to reliably measure influenza-specific IFN-γ as a marker of the cellular immune response using both enzyme-linked immunospot (ELISpot) and enzyme-linked immunosorbent (ELISA) techniques. Here we demonstrate the application of these tools to evaluate cellular immunity in ferrets infected with clinically relevant seasonal H1N1 and H3N2 IAV subtypes at equivalent doses. Using small heparinised blood samples we were able to observe the longitudinal influenza-specific IFN-γ responses of ferrets infected with both seasonal subtypes of IAV and found a notable increase in influenza-specific IFN-γ responses in circulating peripheral blood within 8 days post-infection. Both seasonal strains caused a well-defined pattern of influenza-specific IFN-γ responses in infected ferrets when compared to naïve animals. Additionally, we found that while the influenza specific IFN-γ responses found in peripheral circulating blood were comparable between subtypes, the influenza specific IFN-γ responses found in lung lymphocytes significantly differed. Our results suggest that there is a distinct difference between the ability of the two seasonal influenza strains to establish an infection in the lung of ferrets associated with distinct signatures of acquired immunity.

Animal Models in Influenza Research.

Animal model systems for human and animal influenza virus infection and transmission have been established to address research questions which cannot be addressed using in vitro models. Several animal models have been established, such as mice, guinea pig, ferret, pig, poultry, nonhuman primates, and others. Each animal model has its own strength and weaknesses, which should be taken into consideration to select the appropriate animal model to use. This chapter will describe standard protocols relevant for in vivo experiment, including procedures required prior to the start of the animal experiment and sample processing. The animal models described in this chapter are mice, guinea pigs, ferrets, pigs, and chickens.

Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Low dose influenza virus challenge in the ferret leads to increased virus shedding and greater sensitivity to oseltamivir.

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines.

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection.

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.

Cloning and characterization of chicken IL-10 and its role in the immune response to Eimeria maxima.

We isolated the full-length chicken IL-10 (chIL-10) cDNA from an expressed sequence tag library derived from RNA from cecal tonsils of Eimeria tenella-infected chickens. It encodes a 178-aa polypeptide, with a predicted 162-aa mature peptide. Chicken IL-10 has 45 and 42% aa identity with human and murine IL-10, respectively. The structures of the chIL-10 gene and its promoter were determined by direct sequencing of a bacterial artificial chromosome containing chIL-10. The chIL-10 gene structure is similar to (five exons, four introns), but more compact than, that of its mammalian orthologues. The promoter is more similar to that of Fugu IL-10 than human IL-10. Chicken IL-10 mRNA expression was identified mainly in the bursa of Fabricius and cecal tonsils, with low levels of expression also seen in thymus, liver, and lung. Expression was also detected in PHA-activated thymocytes and LPS-stimulated monocyte-derived macrophages, with high expression in an LPS-stimulated macrophage cell line. Recombinant chIL-10 was produced and bioactivity demonstrated through IL-10-induced inhibition of IFN-gamma synthesis by mitogen-activated lymphocytes. We measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line C.B12) and susceptible (line 15I) chickens during the course of an E. maxima infection. Susceptible chickens showed higher levels of chIL-10 mRNA expression in the spleen, both constitutively and after infection, and in the small intestine after infection than did resistant chickens. These data indicate a potential role for chIL-10 in changing the Th bias during infection with an intracellular protozoan, thereby contributing to susceptibility of line 15I chickens.