Abstract 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.

Author Summary Immunocompromised patients, such as transplant recipients on immune suppressive therapy, are a substantial and gradually expanding patient group. Upon influenza virus infection, these patients clear the virus less efficiently and are more likely to develop severe pneumonia than immunocompetent individuals. Existing antiviral strategies are far from satisfactory for this patient group, as they show limited effectiveness with frequent emergence of antiviral resistance. For ethical and practical reasons antiviral efficacy studies are hard to conduct in these patients. Therefore, we developed an immunocompromised ferret, mimicking an immune suppressive regimen used for solid organ transplant recipients. Upon infection with 2009 pandemic influenza A/H1N1 virus these animals, like immunocompromised patients, develop severe respiratory disease with prolonged virus excretion. Interestingly, all immunocompromised ferrets on oseltamivir therapy excreted oseltamivir resistant viruses (H275Y) within one week after start of treatment. Furthermore, high dose oseltamivir therapy still proved to be partially effective against these oseltamivir resistant viruses. These immunocompromised ferrets provide a useful tool in the development of novel antiviral approaches for immunocompromised patients suffering from influenza.

Citation: van der Vries E, Stittelaar KJ, van Amerongen G, Veldhuis Kroeze EJB, de Waal L, Fraaij PLA, et al. (2013) Prolonged Influenza Virus Shedding and Emergence of Antiviral Resistance in Immunocompromised Patients and Ferrets. PLoS Pathog 9(5): e1003343. https://doi.org/10.1371/journal.ppat.1003343 Editor: Veronika von Messling, Paul-Ehrlich-Institut, Germany Received: November 6, 2012; Accepted: March 21, 2013; Published: May 23, 2013 Copyright: © 2013 van der Vries et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Part of this work was funded by Hoffmann-La Roche Ltd. Hoffmann-La Roche did not participate in any of the work leading to this manuscript. For the immunocompromised ferret model a PCT application has been filed (PCT/EP2012/063497). ADMEO is chief science officer of Viroclinics Biosciences BV, a contract research organisation that collaborates with pharmaceutical companies. PF and AO are financially supported by the Virgo consortium, which is funded by the Dutch government project number FES0908 and the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal's policy and athors have the following conflicts: ADMEO is chief scientific officer of Viroclinics Biosciences BV. Part of this work was funded by Hoffmann-La Roche Ltd. Hoffmann-La Roche did not participate in any of the work leading to this manuscript. For the immunocompromised ferret model a patent is pending (PCT/EP2012/063497). This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

Introduction During the first 12 months of the 2009 influenza A/H1N1 virus (pH1N1) pandemic an estimated 284,000 patients died and hospitalization rates were considerably higher than for seasonal influenza [1]. Although many severe cases were observed in otherwise healthy patients under 50 years of age, most fatal cases during this pandemic were patients belonging to the traditional high risk groups for developing severe disease, like very young children, the elderly and chronically ill patients [2]. In these patients, which in most cases have sub-optimal immune responses, influenza viruses often persists longer and tend to spread more readily into the lower respiratory tract [3], [4], [5], [6]. These observations are in contrast to those in otherwise healthy patients younger than 65 years, for which influenza usually remains a self-limiting upper respiratory tract infection [7], [8]. It has been recognized that every winter season a significant number of immunocompromised patients are admitted to a hospital with influenza [9], [10]. For example, of the total 335 influenza A virus infected patients being diagnosed upon admission to ErasmusMC - a tertiary university hospital - between August 2009 and July 2012, 113 (34%) had an underlying condition that classified them as being immunocompromised [11]. Since immunocompromised patients are more likely to acquire influenza [12], [13], showing relatively high influenza-associated mortality [4], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], effective antiviral prophylaxis and treatment protocols are of crucial importance for these patients. Unfortunately, present antiviral strategies are merely based on clinical trials conducted in otherwise healthy patients [25], since randomized clinical trials in immunocompromised patients are, for both ethical and practical reasons, difficult to perform. Furthermore, the degree and cause of a patient's immunocompromised state is variable and consequently, clinical outcome of infection may vary accordingly. Although antiviral therapy has a documented positive effect on clinical outcome in immunocompromised patients [3], [26], [27], [28], current antiviral strategies are far from satisfying. This may be explained not only by the lack of evidence based strategies adjusted for immunocompromised patients [4], [28], but also by the oral and inhaled administration routes which complicate administration in very young and critically ill patients [29], [30], . Furthermore, since physicians may not consider the diagnosis influenza initially, antiviral therapy is often initiated beyond 48 hours [34], and accompanied by the emergence of an oseltamivir resistant virus [35]. We investigated the incidence of prolonged virus shedding and emergence of antiviral resistance by studying the course of infection of the immunocompromised patients infected with pH1N1 virus treated in our university hospital. These phenomena were studied in more detail in immunocompromised ferrets experimentally infected with pH1N1 virus, that closely mimic immunocompromised patients with influenza. These ferrets all showed prolonged virus shedding and emergence of antiviral resistance. Unexpectedly, the group of immunocompromised ferrets treated with an oseltamivir dose equivalent to a 450 mg dose (twice daily) in humans, had a higher survival rate than similarly untreated animals when infected with an oseltamivir resistant virus.

Discussion Here we show that prolonged influenza virus shedding and the emergence of oseltamivir resistance are two phenomena commonly observed in immunocompromised patients during antiviral therapy. As resistance development is low (19 out of 874 (2.2%)) treated influenza virus infected patients [41], the incidence in immunocompromised patients appears to be considerably higher. Of all 71 immunocompromised patients infected with an influenza A virus in our hospital, at least 16 (23%) (11 pH1N1 and 5 H3N2) showed virus persistence for longer than 2 weeks with 5 of them harbouring oseltamivir resistant virus (5/16) (31%). Because 52% of the included patients were sent home before a virus negative follow-up sample had marked the end of infection, final conclusions on the true incidence of prolonged virus shedding and development of oseltamivir resistant virus cannot be made. However, this observation is in line with previously observed high resistance levels (33%) among paediatric cancer patients [35], and stresses the importance of a thorough evaluation of the currently used antiviral therapies in immunocompromised patients. Since for both logistic and ethical reasons randomized studies are difficult to perform in often critically ill immunocompromised patients, we showed that prolonged influenza virus replication is also a common feature in immunocompromised ferrets. We observed an absence of a rise in body temperature, reduction of lymphocyte proliferation and follicle formation in ferret lymphoid tissues, which are also hallmarks in immunocompromised patients [42] and found a significant reduction of influenza virus specific antibodies in serum. In the 2007/2008 H1N1 virus season an H275Y oseltamivir resistant mutant emerged, which had completely overtaken the circulating virus population by the end of 2008 [43]. This introduction of the H275Y mutation disqualified oseltamivir as the first line antiviral drug. It might happen again with pH1N1 virus if it would also harbour the H275Y mutation without loss of viral fitness. Recent reports on clusters of transmitted pH1N1 H275Y mutant viruses are the first indication that this may not be an unlikely scenario [44], [45]. Early studies on H275Y pH1N1 viral fitness were performed on viruses isolated shortly after the start of the 2009 pandemic. These viruses were found to be at least slightly compromised in their pathogenicity and replication capacity [46], [47], [48], [49]. We also used an early H275Y virus isolate in our experiment [38]. If virus replication is not tempered by adequate immune responses, duration of virus shedding will eventually be restricted by an exhaustion of susceptible host target cells. This then may explain why replication of this apparently less pathogenic virus lasted longest in our immunocompromised ferrets (Figure 3D). Priority should now be given to study the overall fitness of these recent pH1N1 viruses that appear not to be affected by the H275Y change. The use of our immunocompromised ferrets seems to be a very suitable strategy for this purpose, because subtle fitness costs may be amplified in these animals [50]. In our study, immunocompromised ferrets were treated with an oseltamivir dose of 10 mg/kg twice daily. This dose is equivalent to a much higher human dose than the currently recommended dose of 75 mg twice daily [51]. We observed that, for animals infected with either wild type or H275Y mutant virus, high dose oseltamivir treatment was still beneficial. Currently the World Health Organisation recommends switching to zanamivir when dealing with such a resistant virus [52]. However, in the light of our data, discontinuation of oseltamivir therapy may not always be the best strategy. An increased oseltamivir dose for the treatment of immunocompromised patients may be considered as a future antiviral strategy. However, obviously this will need further clinical investigation first. Of note, an increased dose of oseltamivir is well tolerated in humans [53]. We observed lower mortality in the treated animals infected with H275Y mutant virus without an observed difference in virus titers in the upper respiratory tract. In these ferrets, oseltamivir carboxylate plasma levels peaked (C max ) from 3052 ng/ml in 4 hours to 833 ng/ml 12 hours after administration (Table 2 and Figure S1C). These levels were still about 100 and 30 times higher than the 50% inhibitor concentration of an H275Y mutant virus, which is roughly 30 ng/ml (∼100 nM) [54]. It is therefore plausible that oseltamivir therapy reduced H275Y mutant virus titer in the lungs, but not in the upper respiratory tract. This had been observed before and could explain the lower mortality rates in the treated animals [55]. It will therefore be of interest to study penetration of oseltamivir throughout the ferret respiratory tract in more detail, which may be expected to have a direct impact on the effectiveness of the presently recommended human dose in immunocompromised ferrets. In conclusion, both our clinical observations and ferret experiments show that viral clearance cannot be achieved in the immunocompromised host solely by the use of currently used antiviral therapy. Our immunocompromised ferrets may therefore be an excellent alternative to evaluate and explore novel therapeutic and immunization strategies for immunocompromised patients.

Materials and Methods Patient inclusion criteria and diagnostics We identified patients hospitalized in the Erasmus Medical Centre (ErasmusMC), a large (>40,000 admissions in 2011) tertiary university hospital in the Netherlands, with an influenza A virus positive respiratory specimen taken between August 2009 through July 2012. Patients had a prolonged virus infection if the virus could still be detected after 14 days. Virological data, patient immune status and administration of antiviral therapy were obtained by reviewing medical records. Immunosuppression was defined as any of the following: receipt of treatment for any cancer, the use of any immunosuppressive medication to prevent transplant rejection or for management of pulmonary or autoimmune conditions, premature birth and below gestational age or a diagnosis of AIDS. Influenza A virus and the H275Y oseltamivir resistance mutation were detected by reverse transcriptase RT-PCR assays. These assays have been described previously [40], [56]. Informed consent was waived because patient inclusion was performed retrospectively and data were anonymously stored as agreed by the hospital medical ethical board (MEC-2012-463) Viruses used in ferret experiment Two biologically cloned pH1N1 influenza viruses were used in this study. Both viruses were isolated from an oseltamivir treated patient during the first wave of the pandemic in October 2009. Wild type influenza virus A/Netherlands/1715b/2009 (genbank ID code: CY065810) and H275Y mutant virus were isolated from the original quasispecies by co-cultivation of a respiratory sample in a Madin-Darby Canine Kidney (MDCK) cell culture in a single passage [38]. Biological clones were then obtained by 3 additional MDCK passages performed under limiting virus concentrations. As determined by full-genome Sanger sequencing, the mutant virus contained, additional to mutation H275Y in the neuraminidase, an L233M mutation in PB2 and a V541L mutation in HA. Ferrets Animal were housed and experiments were conducted in strict compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). The protocol was approved by the independent animal experimentation ethical review committee from the Netherlands Vaccine Institute (permit number 200900201). All experiments were performed under animal bio-safety level 3 conditions. Animal welfare was observed on a daily basis, and all animal handling was performed under light anaesthesia using a mixture of ketamine and medetomidine to minimize animal suffering. After handling atipamezole was administered to antagonize the effect of medetomidine. All ferrets were eleven-month-old purpose-bred males (body weights between 1562 and 2362 g) and were seronegative for Aleutian disease virus and circulating influenza virus (sub) types A/H1N1, A/H3N2 and B virus. The animals were maintained in standard housing and were transferred to negatively pressured glove boxed on the day immunosuppressive therapy was started. They were provided food ad libidum with commercial food pellets and water. Approximately three to four weeks prior to the experiment a temperature logger (DST micro-T ultra small temperature logger; Star-Oddi, Reykjavik, Iceland) was placed in the peritoneal cavity of the animals. This device recorded the body temperature of the animals every 10 minutes. From day −8 to day −4, an average baseline temperature was recorded for each group of 6 animals. Immunosuppressive, antibiotic and antiviral drugs The following immunosuppressive drugs were used to suppress the immune system of ferrets: Mycophenolate mofetil (MMF) (CellCept, Roche, The Netherlands) powder for infusion, tacrolimus concentrate (5 mg/ml) for infusion (Prograft, Astellas Pharma BV, Leiderdorp, The Netherlands) and prednisolone sodium phosphate (5 mg/ml) oral solution (Hospital Pharmacy, UMCN St Radboud, Nijmegen, The Netherlands). All ferrets received an antibiotic prophylaxis of amoxicillin supplemented with 62.5 mg clavulanic acid (250/62.5 mg per 5 ml) oral suspension (Pharmachemie BV, Haarlem, The Netherlands). Prodrug oseltamivir phosphate, used in the ferret experiments, was kindly provided by Hoffman-La Roche LtD. (Tamiflu, Basel, Switzerland). Other chemicals and reagents Oseltamivir standards for mass spectrometry, oseltamivir phosphate (OS), oseltamivir-d3 (OS-d3), oseltamivir carboxylate (OSC) and oseltamivir carboxylate-d3 (OSC-d3) were purchased from Toronto Research Chemicals (Toronto, Canada). Mycophenolic acid (MPA) standard and internal standards (MPAC) were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands) and from Hoffman-La Roche LtD. respectively. The tacrolimus standards and internal standards (Ascomycin) were purchased from, respectively, Chromsystems and Sigma Aldrich. ULC/MS grade methanol and water containing 0.1% formic acid were obtained from Biosolve (Valkenswaard, the Netherlands). Trichloro acetic acid (TCA) and formic acid (>96%, HCOOH) were obtained from Sigma Aldrich and both were from ACS reagent quality. Administration of the drugs A schematic of the ferret experiment is presented in Figure 2A. Shortly before gavage, drugs were prepared as follows: MMF was dissolved in a 5% glucose solution (Baxter, Unterschleisheim, Germany) to 33 mg/ml. Amoxicillin/clavulanic acid was diluted 5 times in water to obtain a suspension containing 50/12.5 mg/ml amoxicillin/clavulanic acid. Oseltamivir phosphate was dissolved in 5% glucose to 20 mg/ml. These intermediate preparations and the ready-to-use tacrolimus and prednisolone solutions were then used to dose the animals orally and twice daily, as follows: four days before infection, all 6 groups received 10/2.5 mg/kg amoxicillin/clavulanic acid diluted in 5% glucose to a final administration volume of 4 ml/kg. One day later, antibiotic prophylaxis was supplemented to the regimes of groups 2, 3, 5 and 6 with 20 mg/kg MMF, 0.5 mg/kg tacrolimus and 8 mg/kg prednisolone retaining the administration volume of 4 ml/kg. On day 1, 24 hours after infection, therapy of group 3 and 6 was further supplemented with 10 mg/kg oseltamivir phosphate for the remaining 20 days of the experiment. The dose of prednisolone was halved every 7 days from 8 mg/kg in the first to 1 mg/kg in the last week. Virology and serology On day 0, three days after start of immunosuppressive therapy, ferrets were intratracheally infected with 1×104 TCID 50 of wild type (groups 1, 2 and 3) or mutant virus (groups 4, 5 and 6). Each day, pharyngeal and nasal swabs were collected just before administration of the drugs. Swabs were resuspended in 3 ml virus transport medium [57], and aliquots were made and used either directly for online detection of viral RNA by RT-PCR or stored at −80°C for retrospective virus titration. An electron microscopy counted influenza A virus stock was run in parallel to convert RT-PCR cycle threshold (CT) values into a viral particle count. Blood samples for serum and plasma were collected on day 13 after infection. Influenza antibody titers were determined as described previously [58]. Oseltamivir blood plasma levels Oseltamivir and MMF plasma levels and whole blood tacrolimus levels were determined in a pharmacokinetic pilot study. For 4 days, four groups of ferrets (n = 4) received MMF, tacrolimus, or oseltamivir in combination with amoxicillin/clavulanic acid and prednisolone or as the complete cocktail. On day 4, blood was collected from these animals after 0, 10, 20, 30 minutes and 1, 2, 4, 5, 8 and 12 hours after administration of the drugs in order to determine MMF and tacrolimus levels, as described previously [59, unpublished data]. Ferret oseltamivir plasma levels were determined as described previously with some modifications [60]. Calibrators used for the determination of the calibration curve of OS and OSC were prepared from one single stock solution in plasma each containing 50 µg/ml. Calibrators were then prepared by serial dilutions using drug-free plasma. Calibrators for OS and OSC yielded following concentrations: 5000, 1500, 750, 500, 250, 150, 50, 12.5, 2.5 and 0 ng/ml (blank). Aliquots of 50 µl were spiked with 5 µl internal standard solution containing 50 µg/ml OS-d3 and OSC-d3. To calibrators and ferret plasma (K 2 EDTA) samples, 5 µl of a 50% TCA solution (w/v) was added for plasma protein precipitation. Precipitated plasma proteins were removed by centrifugation for 10 minutes at 2000×g at ambient temperature. De-proteinized plasma samples (20 µl) were 2.5 times diluted with ultrapure water and 40 µl of the diluted samples were injected by an auto-sampler (kept at 4°C) into the liquid chromatography mass spectrometry (LC-MS) system. The calibration curves for OS and OSC showed a linear relationship between the SRM peak area of ratios between OS/OS-d3 and OSC/OSC-d3, respectively (OS; r2 = 0.9966 and OSC; r2 = 0.9970). The LLOQ and LOD were determined according to FDA guidelines and were, respectively, 2.5 and 1.0 ng/ml for both OS and OSC. The LC-MS system used was an 4000 API triple quadruple mass spectrometer containing a Turbo V electron spray ion source (ESI) (AB Sciex, Concord, Canada) operating in the positive ionization mode using selected reaction monitoring (SRM) in combination with a Dionex Ultimate 3000 UHPLC system (Amsterdam, the Netherlands) using an Ascentis Express RP-C18 column (100×2.1, 2.7 µm, Supelco, Munich) applying a gradient separation at 30°C. Pathology Samples for histological examination of the tonsils and tracheobronchial lymph nodes were taken to evaluate the immune status and were stored in 10% neutral-buffered formalin. Subsequently, these were routinely processed and embedded in paraffin wax, sectioned at 4 µm and stained with haematoxylin and eosin (HE) for examination by light microscopy. Statistical analysis Data are reported as mean ± standard error of the mean (s.e.m). The P values for comparison of influenza HI antibody titers in figure 2B, virus titers in figure 3 and body weight loss in figure 5C were calculated using Mann-Whitney U test only if at least three animals were remaining in each experimental group. P≤0.05 was considered significant.

Supporting Information Figure S1. Mean steady state (day 4) pharmacokinetics of MMF, tacrolimus and oseltamivir in ferrets. Twice daily, four ferrets were given a cocktail of antibacterial prophylaxis, immune suppressive therapy (a; b) and oseltamivir phosphate (c) for 4 days. On day 4, blood was collected after 0, 10, and 30 minutes and 1, 2, 4, 5, 8 and 12 hours after the final cocktail was administered. Plasma levels of the active form of mycophenolate mofetil (MMF), the metabolite mycophenolic acid (MPA) (a), whole blood tacrolimus levels (b), oseltamivir phosphate (black squares; c) and its metabolite oseltamivir carboxylate (grey squares; c) plasma levels were determined by mass spectrometry. Area under the curve (AUC 0–12 ), peak (C max ) and trough (C 12 ) levels and half-life (t 1/2 ) values are presented in table 2. Data are mean ± s.e.m.. https://doi.org/10.1371/journal.ppat.1003343.s001 (TIF) Figure S2. Ferrets on immune-suppressive therapy show reduced antibody titers. Reduction of serum hemagglutination inhibiting (HI) antibody titers against pH1N1 A/NL/602/2009 virus (a) and more distant viruses A/sw/NL/25/80 (b), A/IT/1443/76 (c), and A/New Jersey/08/76 (d). Individual data points represent antibody titers for each animal and horizontal bars represent the mean titer per group. Body temperature profiles show the absence of fever during the acute stage of infection in ferrets infected with wild type (e) or mutant (f) virus. Animals in this experiment were either immunocompetent (circles), immunocompromised (triangles) or immunocompromised and oseltamivir treated (squares). Data are mean ± s.e.m. Data used for Figure 2B are marked with an asterisk. https://doi.org/10.1371/journal.ppat.1003343.s002 (TIF) Figure S3. Total number of animals positive for replication competent influenza virus in the upper respiratory tract. Influenza virus titers (TCID 50 /ml) were determined in nose and throat swabs daily taken from immunocompetent (blue bars; group 1 and 4) and immunocompromised ferrets (red bars; group 2, 3, 5 and 6). The animals were infected either with wild type (WT; a, b) or mutant virus (H275Y; c, d). Ferrets in groups 3 and 6 were treated with oseltamivir (10 mg/kg twice daily) starting 24 hours after inoculation. https://doi.org/10.1371/journal.ppat.1003343.s003 (TIF)

Acknowledgments We thank Hans van Veen from the Netherlands Vaccine Institute, Ronald Boom, Cindy van Hagen and Willem van Aert from Viroclinics Biosciences BV and Anne van der Linden, Lonneke Leijten, Peter van Run and David van de Vijver from the ErasmusMC virology department, for their great technical assistance. We thank both Annelies Riezebos-Brilman from the department of medical microbiology, division of clinical virology, University of Groningen, University Medical Centre Groningen, The Netherlands and the Dutch national influenza centre for providing patient material from which the influenza biological virus clones were isolated. We thank Prof. Tilanus from ErasmusMC department of surgery and Prof. Van Gelder from the ErasmusMC department of hospital pharmacy for their consultancy regarding immunosuppressive therapy.

Author Contributions Conceived and designed the experiments: EvdV KJS AGV BK MS ADMEO. Performed the experiments: EvdV KJS GvA EJBVK LdW RJM BK. Analyzed the data: EvdV KJS GvA EJBVK LdW PLAF RJM. Contributed reagents/materials/analysis tools: RJM TML BvdN BK. Wrote the paper: EvdV KJS ADMEO.