Edwin J.B. Veldhuis Kroeze

Pathogenesis of influenza-induced acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a fatal complication of influenza infection. In this Review we provide an integrated model for its pathogenesis. ARDS involves damage to the epithelial-endothelial barrier, fluid leakage into the alveolar lumen, and respiratory insufficiency. The most important part of the epithelial-endothelial barrier is the alveolar epithelium, strengthened by tight junctions. Influenza virus targets these epithelial cells, reducing sodium pump activity, damaging tight junctions, and killing infected cells. Infected epithelial cells produce cytokines that attract leucocytes--neutrophils and macrophages--and activate adjacent endothelial cells. Activated endothelial cells and infiltrated leucocytes stimulate further infiltration, and leucocytes induce production of reactive oxygen species and nitric oxide that damage the barrier. Activated macrophages also cause direct apoptosis of epithelial cells. This model for influenza-induced ARDS differs from the classic model, which is centred on endothelial damage, and provides a rationale for therapeutic intervention to moderate host response in influenza-induced ARDS.

Canine Mammary Tumours, an Overview

Canine mammary tumours (CMTs) are the most common neoplasms in intact female dogs. Although the prevalence of these tumours decreases in regions where preventive ovari(ohyster)ectomy is performed, it remains an important disease entity in veterinary medicine. Moreover, treatment options are limited in comparison with human breast cancer. Nevertheless, recent human treatment protocols might have potential in bitches suffering from CMTs.

Prolonged Influenza Virus Shedding and Emergence of Antiviral Resistance in Immunocompromised Patients and Ferrets

Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies.

Multidrug resistant 2009 a/h1n1 influenza clinical isolate with a neuraminidase i223r mutation retains its virulence and transmissibility in ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Influenza virus and endothelial cells: a species specific relationship

Influenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves increased viral tropism for endothelial cells. HPAI virus infections not only cause severe disease in chickens and other terrestrial poultry species but can also spread to humans and back to wild bird populations. Here, we review the role of the endothelium in the pathogenesis of influenza virus infection in wild birds, terrestrial poultry and humans with a particular focus on HPAI viruses. We demonstrate that whilst the endothelium is an important target of virus infection in terrestrial poultry and some wild bird species, in humans the endothelium is more important in controlling the local inflammatory milieu. Thus, the endothelium plays an important, but species-specific, role in the pathogenesis of influenza virus infection.

Pandemic H1N1 vaccine requires the use of an adjuvant to protect against challenge in naïve ferrets

In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naïve ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naïve ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets.

Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals. This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets.

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.

Intranasally administered Endocine™ formulated 2009 pandemic influenza H1N1 vaccine induces broad specific antibody responses and confers protection in ferrets

Influenza is a contagious respiratory disease caused by an influenza virus. Due to continuous antigenic drift of seasonal influenza viruses, influenza vaccines need to be adjusted before every influenza season. This allows annual vaccination with multivalent seasonal influenza vaccines, recommended especially for high-risk groups. There is a need for a seasonal influenza vaccine that induces broader and longer lasting protection upon easy administration. Endocine is a lipid-based mucosal adjuvant composed of endogenous lipids found ubiquitously in the human body. Intranasal administration of influenza antigens mixed with this adjuvant has been shown to induce local and systemic immunity as well as protective efficacy against homologous influenza virus challenge in mice. Here we used ferrets, an established animal model for human influenza virus infections, to further investigate the potential of Endocine as an adjuvant. Intranasal administration of inactivated pandemic H1N1/California/2009 split antigen or whole virus antigen mixed with Endocine induced high levels of serum hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers that were also cross reactive against distant swine viruses of the same subtype. HI and VN antibody titers were already demonstrated after a single nasal immunization. Upon intratracheal challenge with a homologous challenge virus (influenza virus H1N1/The Netherlands/602/2009) immunized ferrets were fully protected from virus replication in the lungs and largely protected against body weight loss, virus replication in the upper respiratory tract and pathological changes in the respiratory tract. Endocine formulated vaccines containing split antigen induced higher HI and VN antibody responses and better protection from body weight loss and virus shedding in the upper respiratory tract than the Endocine formulated vaccine containing whole virus antigen.

Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts

Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies.