Responses In Ferrets Research Articles

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.

Polymer formulated self-amplifying RNA vaccine is partially protective against influenza virus infection in ferrets.

COVID-19 has demonstrated the power of RNA vaccines as part of a pandemic response toolkit. Another virus with pandemic potential is influenza. Further development of RNA vaccines in advance of a future influenza pandemic will save time and lives. As RNA vaccines require formulation to enter cells and induce antigen expression, the aim of this study was to investigate the impact of a recently developed bioreducible cationic polymer, pABOL for the delivery of a self-amplifying RNA (saRNA) vaccine for seasonal influenza virus in mice and ferrets. Mice and ferrets were immunized with pABOL formulated saRNA vaccines expressing either haemagglutinin (HA) from H1N1 or H3N2 influenza virus in a prime boost regime. Antibody responses, both binding and functional were measured in serum after immunization. Animals were then challenged with a matched influenza virus either directly by intranasal inoculation or in a contact transmission model. While highly immunogenic in mice, pABOL-formulated saRNA led to variable responses in ferrets. Animals that responded to the vaccine with higher levels of influenza virus-specific neutralizing antibodies were more protected against influenza virus infection. pABOL-formulated saRNA is immunogenic in ferrets, but further optimization of RNA vaccine formulation and constructs is required to increase the quality and quantity of the antibody response to the vaccine.

Optimization of M2e cassette amino acid composition for the development of universal influenza vaccine

The development of a universal influenza vaccine with a wide spectrum and duration of action is one of the serious public health problems. This study is dedicated to optimization of an immunogen covering the M2e epitopes of influenza A viruses circulating in the natural reservoir, as well as the creation of a prototype of a universal influenza vaccine with subsequent quantitative and qualitative assessment of the induced anti-M2e responses in ferrets.

Distinct higher-order representations of natural sounds in human and ferret auditory cortex.

Little is known about how neural representations of natural sounds differ across species. For example, speech and music play a unique role in human hearing, yet it is unclear how auditory representations of speech and music differ between humans and other animals. Using functional ultrasound imaging, we measured responses in ferrets to a set of natural and spectrotemporally matched synthetic sounds previously tested in humans. Ferrets showed similar lower-level frequency and modulation tuning to that observed in humans. But while humans showed substantially larger responses to natural vs. synthetic speech and music in non-primary regions, ferret responses to natural and synthetic sounds were closely matched throughout primary and non-primary auditory cortex, even when tested with ferret vocalizations. This finding reveals that auditory representations in humans and ferrets diverge sharply at late stages of cortical processing, potentially driven by higher-order processing demands in speech and music.

Intranasal powder live attenuated influenza vaccine is thermostable, immunogenic, and protective against homologous challenge in ferrets

Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and elicit robust immunity. However, some LAIVs require reconstitution and cold-chain requirements restrict storage and distribution of all influenza vaccines. We generated a dry-powder, thermostable LAIV (T-LAIV) using Preservation by Vaporization technology and assessed the stability, immunogenicity, and efficacy of T-LAIV alone or combined with delta inulin adjuvant (Advax™) in ferrets. Stability assays demonstrated minimal loss of T-LAIV titer when stored at 25 °C for 1 year. Vaccination of ferrets with T-LAIV alone or with delta inulin adjuvant elicited mucosal antibody and robust serum HI responses in ferrets, and was protective against homologous challenge. These results suggest that the Preservation by Vaporization-generated dry-powder vaccines could be distributed without refrigeration and administered without reconstitution or injection. Given these significant advantages for vaccine distribution and delivery, further research is warranted.

AS03-adjuvanted H7N9 inactivated split virion vaccines induce cross-reactive and protective responses in ferrets

Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model. Serological analyses demonstrated that homologous hemagglutination inhibition and microneutralization antibody titers were detectable in the ferrets after the first immunization with the AS03-adjuvanted vaccines that were further boosted by the second immunization. In addition, heterologous antibody titers against older H7 subtype viruses of the North American lineage (H7N7, H7N3) and newer H7 subtype viruses of the Eurasian lineage (H7N9) were detected in the animals receiving the AS03-adjuvanted vaccines. Animals receiving two immunizations of the AS03-adjuvanted vaccines were protected from weight loss and fever in the homologous challenge study and had no detectable virus in throat or lung samples. In addition, microscopic examination post-challenge showed animals immunized with the AS03-adjuvanted vaccines had the least signs of lung injury and inflammation, consistent with the greater relative efficacy of the adjuvanted vaccines. In conclusion, this study demonstrated that the AS03-adjuvanted H7N9 vaccines elicited high levels of homologous and heterologous antibodies and protected against H7N9 virus damage post-challenge.

Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.

Influenza-specific ex vivo immune responses in ferrets commensurate with number of vaccinations despite decreased protection for repeat vaccines

Abstract There is controversy in the literature whether consecutive year influenza vaccinations lead to decreased vaccine efficacy. Evidence for this effect is strongest in the context of repeated H3N2 vaccinations. As recently reported (NPJ Vaccines 4:28 (2019)), we recreated this phenomenon in the ferret model of influenza. Ferrets exposed to consecutive year vaccinations had significantly increased weight loss, viral shedding and fever after A/Hong Kong/4801/2014 (HK4801) virus challenge than ferrets only exposed to the 2017–2018 vaccine. One possible explanation for this phenomenon is that the repeated vaccination induces non-protective antibodies against influenza virus, which might include low avidity antibodies, non-neutralizing antibodies, and antibodies that otherwise do not elicit a protective immune response. Ferrets that received repeated vaccination had higher avidity antibodies to HK4801 HA at Day 2 and 14 post-infection than did the other two groups. Preliminary data do not support increased levels of HA stem-binding antibodies in repeat vaccinees’ serum. NA and NP are two additional viral proteins expressed on the cell surface, with greater or lesser evidence for their role in protective antibody responses, respectively. Serum reactivity to both proteins correlated with the number of vaccinations. Together, these results do not support a model of increased non-protective antibodies after repeat vaccination. Furthermore, the breadth of the T cell response against H3N2 viruses from different clades was similar between both vaccinated groups. Ongoing studies continue to explore the mechanism for the decreased protection observed in the repeat vaccination group compared with the single year vaccination group.

Potential Antigenic Mismatch of the H3N2 Component of the 2019 Southern Hemisphere Influenza Vaccine.

Here, we find that the egg-adapted H3N2 component of the 2019 Southern Hemisphere influenza vaccine elicits an antibody response in ferrets that is highly focused on antigenic site A of hemagglutinin. This is potentially problematic as most H3N2 viruses currently circulating in the Southern Hemisphere possess antigenic site A substitutions.

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.

Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection.

Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.

Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets.

The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound "avian-like" α2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain "human-like" α2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with ≥14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against ≥15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness.IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind "avian-like" sialic acids, although several isolates also displayed binding to "human-like" sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain.

Novel hemagglutinin nanoparticle influenza vaccine with Matrix-M™ adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted A(H3N2) subtypes.

Influenza viruses frequently acquire mutations undergoing antigenic drift necessitating annual evaluation of vaccine strains. Highly conserved epitopes have been identified in the hemagglutinin (HA) head and stem regions, however, current influenza vaccines induce only limited responses to these conserved sites. Here, we describe a novel seasonal recombinant HA nanoparticle influenza vaccine (NIV) formulated with a saponin-based adjuvant, Matrix-M™. NIV induced hemagglutination inhibition (HAI) and microneutralizing (MN) antibodies against a broad range of influenza A(H3N2) subtypes. In a comparison of NIV against standard-dose and high-dose inactivated influenza vaccines (IIV and IIV-HD, respectively) in ferrets NIV elicited HAI and MN responses exceeding those induced by IIV-HD against homologous A(H3N2) by 7 fold, A(H1N1) by 26 fold, and B strain viruses by 2 fold. NIV also induced MN responses against all historic A/H3N2 strains tested, spanning more than a decade of viral evolution from the 2000-2017 influenza seasons whereas IIV and IIV-HD induced HAI and MN responses were largely directed against the homologous A(H3N2), A(H1N1), and B virus strains. NIV induced superior protection compared to IIV and IIV-HD in ferrets challenged with a homologous or 10-year drifted influenza A(H3N2) strain. HAI positive and HAI negative neutralizing monoclonal antibodies derived from mice immunized with NIV were active against homologous and drifted influenza A(H3N2) strains. Taken together these observations suggest that NIV can induce responses to one or more highly conserved HA head and stem epitopes and result in highly neutralizing antibodies against both homologous and drift strains.

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.

Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest.

Eyedrop Vaccination Induced Systemic and Mucosal Immunity against Influenza Virus in Ferrets.

We investigated eyedrop vaccination (EDV) in pre-clinical development for immunological protection against influenza and for potential side effects involving ocular inflammation and the central nervous system (CNS). Live attenuated influenza EDV, CA07 (H1N1), PZ-4 (H1N2) and Uruguay (H3N2), induced both systemic and mucosal virus-specific antibody responses in ferrets. In addition, EDV resulted in a clinically significant protection against viral challenge, and suppression of viral replication in nasal secretion and lung tissue. Regarding safety, we found that administered EDV flow through the tear duct to reach the base of nasal cavity, and thus do not contact the olfactory bulb. All analyses for potential adverse effects due to EDV, including histological and functional examinations, did not reveal significant side effects. On the basis of these findings, we propose that EDV as effective, while being a safe administration route with minimum local side effects, CNS invasion, or visual function disturbance.

Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans

The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines.

Acetylcholinesterase inhibitor modifications: a promising strategy to delay the progression of Alzheimer's disease.

Alzheimer's disease (AD), a fatal, progressive, neurodegenerative disorder, is the most common cause of old-age dementia, accounting for 50–75% of dementia patients. Early stages of AD are marked by vocabulary shrinkage, spatial disorientation, depression, apraxia, and deterioration of recent forms of declarative memory. In course of time, the patients require close supervision due to the loss of cognitive and functional abilities, and at the terminal stages of the disease, all forms of memory are severely impaired with the patients needing nursing home care (World Alzheimer Report, 2013).

Cross-protection against H7N9 influenza strains using a live-attenuated H7N3 virus vaccine

In 2013, avian H7N9 influenza viruses were detected infecting people in China resulting in high mortality. Influenza H7 vaccines that provide cross-protection against these new viruses are needed until specific H7N9 vaccines are ready to market. In this study, an available H7N3 cold-adapted, temperature sensitive, live attenuated influenza vaccine (LAIV) elicited protective immune responses in ferrets against H7N9 viruses. The H7N3 LAIV administered alone (by intranasal or subcutaneous administration) or in a prime-boost strategy using inactivated H7N9 virus resulted in high HAI titers and protected 100% of the animals against H7N9 challenge. Naïve ferrets passively administered immune serum from H7N3 LAIV infected animals were also protected. In contrast, recombinant HA protein or inactivated viruses did not protect ferrets against challenge and elicited lower antibody titers. Thus, the H7N3 LAIV vaccine was immunogenic in healthy seronegative ferrets and protected these ferrets against the newly emerged H7N9 avian influenza virus.

RNA-Based Vaccination: Sending a Strong Message

Nucleic acid–based vaccines (NAVs) for induction of antigen-specific immunity have reemerged as important tools in our vaccine/immune therapeutic arsenal. DNA technology appears to more than hold its own alongside viral vectored systems, particularly when the focus is on driving robust T-cell immunity in the clinic.1 By contrast, studies of vaccines based on RNA in small animals have been disappointing. Instability of the mRNA and manufacturing problems further weakened interest in this platform. However, just as good wine improves with age, cutting-edge technologies can take time to mature, and direct injection of RNA for immunization has recently been shown to be sufficiently improved. Support for RNA delivery as a more effective means of vaccination comes from several very recent reports.2,3,4