Evaluation of Commercial Myxomatosis Vaccines against Recombinant Myxoma Virus (ha-MYXV) in Iberian Hare and Wild Rabbit.

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains.

A broad spectrum HVT-H5 avian influenza vector vaccine which induces a rapid onset of immunity.

Current methods to combat highly pathogenic avian influenza (HPAI) outbreaks in poultry rely on stamping out and preventive culling, which can lead to high economic losses and invoke ethical resistance. Emergency vaccination could be an alternative as vaccination is one of the most efficient and cost-effective measures to protect poultry from HPAI infection, preventing spreading to other poultry and greatly reducing the potential transmission to humans. Current conventional inactivated AI vaccines may be useful for combating AI outbreaks, but do not fulfil all targets of an ideal AI vaccine, including mass applicability and rapid onset of immunity. We aimed to further investigate the potential of Herpesvirus of Turkeys (HVT) as a vector containing a recombinant H5 hemagglutinin of HPAI H5N1. This HVT-H5 vector was analysed in vitro, tested for onset of immunity against AI challenge, breadth of protection, reduction of virus shedding, and induction of both antibody and cellular responses in SPF layers or broiler chicks containing maternal derived antibodies (MDA+). In SPF layers HVT-H5 provided full protection to lethal challenges with 4 antigenically diverse HPAI H5N1 strains from 2 weeks post vaccination (w.p.v.), while in MDA+ birds full protection was provided from 3 w.p.v. to homologous challenge. Also shedding of challenge virus was reduced in both SPF and MDA+ birds. HVT-H5 induced a protective HI titre (≥4) to 11 HPAI H5N1 strains at 3 w.p.v. in 3-week-old SPF layers and to HPAI H5N8 A/ch/Neth/14015531/2014. Besides inducing a protective antibody response HVT-H5 also induced an influenza-specific T cell response. This data demonstrates that HVT-H5 vaccine appears to fulfil many of the criteria for an ideal AI vaccine including early onset of immunity, a broad protection, reduced virus shedding, protection in presence of AI-MDA and could be a useful tool in the combat of AI outbreaks worldwide.

Protection against the New Equine Influenza Virus Florida Clade I Outbreak Strain Provided by a Whole Inactivated Virus Vaccine.

Equine influenza virus (EIV) is a major cause of respiratory disease in horses. Vaccination is an effective tool for infection control. Although various EIV vaccines are widely available, major outbreaks occurred in Europe in 2018 involving a new EIV H3N8 FC1 strain. In France, it was reported that both unvaccinated and vaccinated horses were affected despite >80% vaccination coverage and most horses being vaccinated with a vaccine expressing FC1 antigen. This study assessed whether vaccine type, next to antigenic difference between vaccine and field strain, plays a role. Horses were vaccinated with an ISCOMatrix-adjuvanted, whole inactivated virus vaccine (Equilis Prequenza) and experimentally infected with the new FC1 outbreak strain. Serology (HI), clinical signs, and virus shedding were evaluated in vaccinated compared to unvaccinated horses. Results showed a significant reduction in clinical signs and a lack of virus shedding in vaccinated horses compared to unvaccinated controls. From these results, it can be concluded that Equilis Prequenza provides a high level of protection to challenge with the new FC1 outbreak strain. This suggests that, apart from antigenic differences between vaccine and field strain, other aspects of the vaccine may also play an important role in determining field efficacy.

Determining Equine Influenza Virus Vaccine Efficacy-The Specific Contribution of Strain Versus Other Vaccine Attributes.

Vaccination is an effective tool to limit equine influenza virus (EIV H3N8) infection, a contagious respiratory disease with potentially huge economic impact. The study assessed the effects of antigenic change on vaccine efficacy and the need for strain update. Horses were vaccinated (V1 and V2) with an ISCOMatrix-adjuvanted, whole inactivated virus vaccine (Equilis Prequenza, group 2, FC1 and European strains) or a carbomer-adjuvanted, modified vector vaccine (ProteqFlu, group 3, FC1 and FC2 HA genes). Serology (SRH, HI, VN), clinical signs and viral shedding were assessed in comparison to unvaccinated control horses. The hypothesis was that group 2 (no FC2 vaccine strain) would be less well protected than group 3 following experimental infection with a recent FC2 field strain (A/equi-2/Wexford/14) 4.5 months after vaccination. All vaccinated horses had antibody titres to FC1 and FC2. After challenge, serology increased more markedly in group 3 than in group 2. Vaccinated horses had significantly lower total clinical scores and viral shedding. Unexpectedly, viral RNA shedding was significantly lower in group 2 than in group 3. Vaccination induced protective antibody titres to FC1 and FC2 and reduced clinical signs and viral shedding. The two tested vaccines provided equivalent protection against a recent FC2 EIV field strain.

Novel Trivalent Vectored Vaccine for Control of Myxomatosis and Disease Caused by Classical and a New Genotype of Rabbit Haemorrhagic Disease Virus.

Myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) are the major causes of lethal viral diseases in the European rabbit. In 2010, a new RHDV genotype (RHDV2) emerged in the field that had limited cross-protection with the classical RHDV (RHDV1). For optimal protection of rabbits and preventing spread of disease, a vaccine providing protection against all three key viruses would be ideal. Therefore, a novel trivalent myxoma vectored RHDV vaccine (Nobivac Myxo-RHD PLUS) was developed similar to the existing bivalent myxoma vectored RHDV vaccine Nobivac Myxo-RHD. The new vaccine contains the Myxo-RHDV1 strain already included in Nobivac Myxo-RHD and a similarly produced Myxo-RHDV2 strain. This paper describes several key safety and efficacy studies conducted for European licensing purposes. Nobivac Myxo-RHD PLUS showed to be safe for use in rabbits from five weeks of age onwards, including pregnant rabbits, and did not spread from vaccinated rabbits to in-contact controls. Furthermore, protection to RHDV1 and RHDV2 was demonstrated by challenge, while the serological response to MV was similar to that after vaccination with Nobivac Myxo-RHD. Therefore, routine vaccination with Nobivac Myxo-RHD PLUS can prevent the kept rabbit population from these major viral diseases.

Transcriptomics in lung tissue upon respiratory syncytial virus infection reveals aging as important modulator of immune activation and matrix maintenance.

Aging poses an increased risk of severe infection by respiratory syncytial virus (RSV). The many different biological pathways comprising the response to infection in lungs that are influenced by aging are complex and remain to be defined more thoroughly. Towards finding new directions in research on aging, we aimed to define biological pathways in the acute response to RSV that are affected in the lungs by aging. We therefore profiled the full transcriptome of lung tissue of mice prior to and during RSV infection both at young and old age. In the absence of RSV, we found aging to downregulate genes that are involved in constitution of the extracellular matrix. Moreover, uninfected old mice showed elevated expression of pathways that resemble injury, metabolic aberrations, and disorders mediated by functions of the immune system that were induced at young age only by an exogenous trigger like RSV. Furthermore, infection by RSV mounted stronger activation of anti-viral type-I interferon pathways at old age. Despite such exaggerated anti-viral responses, old mice showed reduced control of virus. Altogether, our findings emphasize important roles in aging-related susceptibility to respiratory disease for extracellular matrix dysfunctions and dysregulated immune activation in lungs.

Genetic versus antigenic differences among highly pathogenic H5N1 avian influenza A viruses: Consequences for vaccine strain selection.

Highly pathogenic H5N1 avian influenza A viruses display a remarkable genetic and antigenic diversity. We examined to what extent genetic distances between several H5N1 viruses from different clades correlate with antigenic differences and vaccine performance. H5-specific antisera were generated, and cross-reactivity and antigenic distances between 12 different viruses were determined. In general, antigenic distances increased proportional to genetic distances although notable exceptions were observed. Antigenic distances correlated better with genetic variation in 27 selected, antigenically-relevant H5 residues, than in the complete HA1 domain. Variation in these selected residues could accurately predict the antigenic distances for a novel H5N8 virus. Protection provided by vaccines against heterologous H5N1 challenge viruses indicated that cross-protection also correlates better with genetic variation in the selected antigenically-relevant residues than in complete HA1. When time is limited, variation at these selected residues may be used to accurately predict antigenic distance and vaccine performance.

Impaired immune response to vaccination against infection with human respiratory syncytial virus at advanced age.

UNLABELLED: Elderly humans are prone to severe infection with human respiratory syncytial virus (HRSV). The aging of today's human population warrants the development of protective vaccination strategies aimed specifically at the elderly. This may require special approaches due to deteriorating immune function. To design and test vaccination strategies tailored to the elderly population, we need to understand the host response to HRSV vaccination and infection at old age. Moreover, the preclinical need for testing of candidate vaccines requires translational models resembling susceptibility to the (unadapted) human pathogen. Here, we explored the effects of aging on immunity and protection induced by a model HRSV vaccine candidate in a translational aging model in cotton rats (Sigmodon hispidus) and examined possibilities to optimize vaccination concepts for the elderly. We immunized young and aged cotton rats with a live-attenuated recombinant HRSV vaccine candidate and analyzed the induced immune response to and protection against challenge with HRSV. In old cotton rats, HRSV infection persisted longer, and vaccination induced less protection against infection. Aged animals developed lower levels of vaccine-induced IgG, virus-neutralizing serum antibodies, and IgA in lungs. Moreover, booster responses to HRSV challenge were impaired in animals vaccinated at an older age. However, increased dose and reduced attenuation of vaccine improved protection even in old animals. This study shows that cotton rats provide a model for studying the effects of aging on the immune response to the human respiratory pathogen HRSV and possibilities to optimize vaccine concepts for the elderly. IMPORTANCE: HRSV infection poses a risk for severe disease in the elderly. The aging of the population warrants increased efforts to prevent disease at old age, whereas HRSV vaccines are only in the developmental phase. The preclinical need for testing of candidate human vaccines requires translational models resembling susceptibility to the natural human virus. Moreover, we need to gain insight into waning immunity at old age, as this is a special concern in vaccine development. In this study, we explored the effect of age on protection and immunity against an experimental HRSV vaccine in aged cotton rats (Sigmodon hispidus), a rodent species that provides a model representing natural susceptibility to human viruses. Older animals generate fewer antibodies upon vaccination and require a higher vaccine dose for protection. Notably, during the early secondary immune response to subsequent HRSV infection, older animals showed less protection and a slower increase of the virus-neutralizing antibody titer.

Chicken dendritic cells are susceptible to highly pathogenic avian influenza viruses which induce strong cytokine responses.

Infection with highly pathogenic avian influenza (HPAI) in birds and mammals is associated with severe pathology and increased mortality. We hypothesize that in contrast to low pathogenicity avian influenza (LPAI) infection, HPAI infection of chicken dendritic cells (DC) induces a cytokine deregulation which may contribute to their highly pathogenic nature. Infection of DC with LPAI H7N1 and H5N2 resulted in viral RNA and NP expression without increase in time, in contrast to HPAI H7N1 and H5N2 mRNA expression. No increase in IFN mRNA was detected after infection with LPAI, but after LPAI H5N2, and not LPAI H7N1, infection the level of bioactive IFNα/ß significantly increased. After HPAI H7N1 and H5N2 infection, significant increases in IL-8, IFN-α, IFN-γ mRNA expression and in TLR1, 3, and 21 mRNA were observed. This enhanced activation of DC after HPAI infection may trigger deregulation of the immune response as seen during HPAI infection in chickens.

Identification of novel avian influenza virus derived CD8+ T-cell epitopes.

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.

Reduced immune reaction prevents immunopathology after challenge with avian influenza virus: a transcriptomics analysis of adjuvanted vaccines.

To gain more insight in underlying mechanisms correlating to protection against avian influenza virus (AIV) infection, we investigated correlates of protection after AIV H9N2 infection and studied the contribution of different adjuvants to a protective response at host transcriptional level. One-day-old chickens were immunised with inactivated H9N2 supplemented with w/o, Al(OH)(3), CpG or without adjuvant. Two weeks later, birds were homologously challenged and at 1-4 days post challenge (d.p.c.) trachea and lung were collected. Birds immunised with H9N2+w/o or H9N2+Al(OH)(3) were protected against challenge infection and had lower viral RNA expression, less immune related genes induced after challenge, a lower amplitude of change of gene expression and smaller cellular influxes compared to the higher and prolonged gene expression in unprotected birds. We show that a limited number of differentially expressed genes correlates with reduced immune activation and subsequently reduced immunopathology after challenge with AIV.

Early host responses to avian influenza A virus are prolonged and enhanced at transcriptional level depending on maturation of the immune system.

Newly hatched chickens are more susceptible to infectious diseases than older birds because of an immature immune system. The aim of this study was to determine to what extent host responses to avian influenza virus (AIV) inoculation are affected by age. Therefore, 1- and 4-week (wk) old birds were inoculated with H9N2 AIV or saline. The trachea and lung were sampled at 0, 8, 16 and 24h post-inoculation (h.p.i.) and gene expression profiles determined using microarray analysis. Firstly, saline controls of both groups were compared to analyse the changes in gene profiles related to development. In 1-wk-old birds, higher expression of genes related to development of the respiratory immune system and innate responses were found, whereas in 4-wk-old birds genes were up regulated that relate to the presence of higher numbers of leukocytes in the respiratory tract. After inoculation with H9N2, gene expression was most affected at 16 h.p.i. in 1-wk-old birds and at 16 and 24h.p.i. in 4-wk-old birds in the trachea and especially in the lung. In 1-wk-old birds less immune related genes including innate related genes were induced which might be due to age-dependent reduced functionality of antigen presenting cells (APC), T cells and NK cells. In contrast cytokine and chemokines gene expression was related to viral load in 1-wk-old birds and less in 4-wk-old birds. Expression of cellular host factors that block virus replication by interacting with viral factors was independent of age or tissue for most host factors. These data show that differences in development are reflected in gene expression and suggest that the strength of host responses at transcriptional level may be a key factor in age-dependent susceptibility to infection, and the cellular host factors involved in virus replication are not.

Transcriptional expression levels of chicken collectins are affected by avian influenza A virus inoculation.

Mammalian collectins have been found to play an important role in the defense against influenza A virus H9N2 inoculation, but for chicken collectins this has not yet been clarified. The aim of this study was to determine the effect of avian influenza A virus (AIV) inoculation on collectin gene expression in the respiratory tract of chickens and whether this was affected by age. For this purpose 1- and 4-week-old chickens were inoculated intratracheally with PBS or H9N2 AIV. Chickens were killed at 0, 8, 16 and 24h post-inoculation and trachea and lung were harvested for analysis. Viral RNA expression and mRNA expression of chicken collectins 1 and 2 (cCL-1 and cCL-2), chicken lung lectin (cLL) and chicken surfactant protein A (cSP-A) were determined using real-time quantitative RT-PCR. In lung, a decrease in mRNA expression of cCL-2, cLL and cSP-A after inoculation with H9N2 was seen in both 1- and 4-week-old birds, although at different time points, while in trachea changes were only seen in 4-week-old birds and expression was increased. Moreover, collectin expression correlated with viral RNA expression in lung of 1-week-old birds. These results suggest that both age and location in the respiratory tract affect changes in collectin mRNA expression after inoculation with H9N2 and indicate a possible role for collectins in the host response to AIV in the respiratory tract of chickens.

Differential gene-expression and host-response profiles against avian influenza virus within the chicken lung due to anatomy and airflow.

Sampling the complete organ instead of defined parts might affect analysis at both the cellular and transcriptional levels. We defined host responses to H9N2 avian influenza virus (AIV) in trachea and different parts of the lung. Chickens were spray-inoculated with either saline or H9N2 AIV. Trachea and lung were sampled at 1 and 3 days post-inoculation (p.i.) for immunocytochemistry, real-time quantitative RT-PCR and gene-expression profiling. The trachea was divided into upper and lower parts and the lung into four segments, according to anatomy and airflow. Two segments contained the primary and secondary bronchi, cranial versus caudal (parts L1 and L3), and two segments contained the tertiary bronchi, cranial versus caudal (parts L2 and L4). Between the upper and lower trachea in both control and infected birds, minor differences in gene expression and host responses were found. In the lung of control birds, differences in anatomy were reflected in gene expression, and in the lung of infected birds, virus deposition enhanced the differences in gene expression. Differential gene expression in trachea and lung suggested common responses to a wide range of agents and site-specific responses. In trachea, site-specific responses were related to heat shock and lysozyme activity. In lung L1, which contained most virus, site-specific responses were related to genes involved in innate responses, interleukin activity and endocytosis. Our study indicates that the anatomy of the chicken lung must be taken into account when investigating in vivo responses to respiratory virus infections.

Cellular host transcriptional responses to influenza A virus in chicken tracheal organ cultures differ from responses in in vivo infected trachea.

In this study a viral infection of a tissue culture model system was compared to an in vivo infection, which is of importance to gauge the utility of the model system. The aim was to characterize early immune responses induced by avian influenza virus using tracheal organ cultures (TOC) as a model system. First, the in vitro system was optimized to ensure that the host transcription responses were only influenced by virus infection and not by differences in viral load. Upper and lower trachea both could be used in the cultures because the virus load was the same. Cilia motility was not affected in non-infected TOC and only slightly in infected TOC at 24h post-inoculation. Gene expression profiles of early immune responses were analyzed in in vitro infected TOC, and were compared to the responses found in in vivo infected trachea. The gene expression profile in infected TOC suggested the up regulation of innate anti-viral responses that were triggered by attachment, entry and uptake of virus leading to several signalling cascades including NF-kappaB regulation. Genes associated with IFN mediated responses were mainly type I IFN related. Overlapping gene expression profiles between non-infected and infected TOC suggested that tissue damage during excision induced wound healing responses that masked early host responses to the virus. These responses were confirmed by real-time quantitative RT-PCR showing up regulation of IL-1beta and IL-6. Microarray analysis showed that gene expression profiles of infected and non-infected TOC had a large overlap. This overlap contained many immune-related genes associated with inflammatory responses, apoptosis and immune system process and development. Infected TOC and in vivo infected trachea shared few significantly differentially expressed genes. The gene expression profile of infected TOC contained fewer genes which were expressed at reduced amplitude of change. Genes that were common between TOC and trachea were associated with early immune responses likely triggered by virus attachment and entry. Most of the genes were associated with IFN-mediated responses, mainly type I IFN related. Our study implicates that although the TOC model is suitable for culturing of virus and lectin or virus binding studies, it is not suitable for measuring early immune responses upon viral infection at host transcriptional level.

Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus.

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to alpha-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity.