INFLUENZA VIRUS AND TYPE 1 DIABETES

Abstract

Type 1 diabetes mellitus is characterized by loss of pancreatic insulin-producing beta cells, resulting in insulin deficiency. The usual cause of this beta cell loss is autoimmune destruction. The inventors provide the first evidence of a causal link between influenza virus infection and the development of type 1 diabetes and/or pancreatitis. This causal link between infection and type 1 diabetes and/or pancreatitis provides various therapeutic, prophylactic and diagnostic opportunities.

Claims

1-20. (canceled)

21. A method of treating type 1 diabetes in a patient comprising: selecting the patient in need of treatment for type 1 diabetes, and vaccinating the patient with an immunogenic composition, wherein the vaccination of the patient prevents or reduces the severity of influenza infection, thereby reducing the effects of the influenza infection on the type 1 diabetes.

22. The method of claim 21, further comprising treating the patient with at least one of the following treatments selected from the group consisting of islet transplantation, transplantation of beta cell precursors, and stem cells.

23. The method of claim 21, wherein the immunogenic composition comprises an adjuvant.

24. The method of claim 23, wherein the adjuvant is MF59.

25. The method of claim 21, wherein the subject is a child.

26. The method of claim 21, wherein levels of CXCL9/MIG are lowered following vaccination.

27. The method of claim 21, wherein levels of CXCL10/IP-10 are lowered following vaccination.

28. The method of claim 21, wherein levels of CCL5/RANTES, CCL4/MIP1b, CXCL1/Groa, CXCL8/IL8, TNFa, and IL-6 are lowered following vaccination.

29. A method of treating pancreatitis in a patient comprising: selecting the patient in need of treatment for pancreatitis, and vaccinating the patient with an immunogenic composition, wherein the vaccination of the patient prevents or reduces the severity of influenza infection, thereby reducing the effects of the influenza infection on the pancreatitis.

30. The method of claim 29, further comprising treating the patient with at least one of the following treatments selected from the group consisting of islet transplantation, transplantation of beta cell precursors, and stem cells.

31. The method of claim 29, wherein the immunogenic composition comprises an adjuvant.

32. The method of claim 31, wherein the adjuvant is MF59.

33. The method of claim 29, wherein the subject is a child.

34. The method of claim 29, wherein levels of CXCL9/MIG are lowered following vaccination.

35. The method of claim 29, wherein levels of CXCL10/IP-10 are lowered following vaccination.

36. The method of claim 29, wherein levels of CCL5/RANTES, CCL4/MIP1b, CXCL1/Groa, CXCL8/IL8, TNFa, and IL-6 are lowered following vaccination.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0160] FIGS. 1A, 1B, and 1C show glucose and lipase plasmatic concentrations for groups A (receiving H7N1 A/turkey/Italy/3675/1999, FIG. 1A), B (receiving H7N3A/turkey/Italy/2962/2003, FIG. 1B) and K (control, FIG. 1C). ID: identification number; n.d.: not done; eut: euthanized in order to collect the samples for histology and immunohistochemistry at designated days post-infection or due to the end of the experiment; columns highlighted in dark grey: days in which only subjects with high lipase concentration were tested with Glucocard strips (upper limit 34 mmol/L); columns highlighted in light grey: particularly relevant data.

[0161] FIGS. 2A and 2B show Kaplan-Meier analyses for the appearance of hyperlipasemia (FIG. 2A) and hyperglycaemia (FIG. 2B) (plasma glucose >27.78 mmol/L,) between the mock, H7N1 and H7N3 infected turkeys. Differences were tested using the log rank statistic. Bar graphs: frequency of events in relation to hyperlipasemia, hyperglycaemia and viraemia.

[0162] FIG. 3 shows a turkey pancreas section (normal tissue). Acinar cells containing zymogen granules in their cytoplasm are evident, associated with two nests of normal islet cells and a ductal structure.

[0163] FIG. 4 shows a turkey pancreas section 7 days post infection. Diffuse and severe necrosis of acinar cells (arrows) with severe inflammatory infiltrate (*).

[0164] FIG. 5 shows a turkey pancreas section. Most of the pancreas is replaced by foci of lymphoid nodules and fibrous connective tissue and lymphoid nodules with some ductular proliferation.

[0165] FIG. 6 shows a turkey pancreas section 4 days post infection. Immunohistochemistry for avian influenza nucleoprotein (NP). Positive nuclei and cytoplasm are evident in necrotic acinar cells and in the ductal epithelium.

[0166] FIGS. 7A, 7B, 7C, and 7D show replication kinetics in pancreatic cell lines of A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/2005 (H3N2) in hCM and HPDE6 cells. hCM and HPDE6 cells were infected with each virus at an MOI=0.001. At 24, 48 and 72 hours post-infection, supernatants from three infected and one mock-infected control well were harvested for virus isolation and qRRT-PCR analysis. FIG. 7A shows virus Isolation results of H1N1 in hCM and HPDE6. FIG. 7B shows qRRT-PCR results of H1N1 in hCM and HPDE6. FIG. 7C shows virus Isolation results of H3N2 in hCM and HPDE6. FIG. 7D shows qRRT-PCR results of H3N2 in hCM HPDE6. All results represent means plus standard deviations of three independent experiments.

[0167] FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, and 8H show Western blot analyses of H1N1 (FIGS. 8A and 8B) and H3N2 (FIGS. 8E and 8F) influenza virus NP expression (56KDa) in hCM and HPDE6 cells. Samples were collected before infection (t0) and 24 (t24), 48 (t48) and 72 (t72) hours post-infection. Beta-actin (42 KDa) was used as loading control in order to assure that the same amount of proteins was tested for each sample (FIGS. 8C, 8D, 8G, and 8H).

[0168] FIGS. 9A, 9B, 9C, and 9D show nuclear staining of HPDE6 negative control (20X) (FIG. 9A). Cells were DAPI stained to reveal bound to DNA and with Evans Blue as contrast. FIG. 9B shows HPDE6 at 24 h post-infection (20X). Influenza virus NP protein derived from viral infection was observed (center of image). FIG. 9C shows HCM negative control. FIG. 9D shows hCM at 24 hours post-infection (20X), Influenza virus NP protein derived from viral infection was observed as brightly coloured cells in the center of the image.

[0169] FIGS. 10A, 10B, 10C, and 10D shows RRT-PCR data for M gene in human pancreatic islets: Two-way quadratic prediction plot with CIs (confidence interval) for RRT-Real time Ct values obtained from H1N1 (FIGS. 10A and 10C) and H3N2 in pancreatic islets (FIGS. 10B and 10D) 4.810.sup.3 PFU/well pancreatic islet cell infection. For each virus are represented the Ct trend in pancreatic islet pellets and supernatants from the day of infection (to) until day 10 (t.sub.5) in presence (first column) or absence (second column) of TPCK and as an average of the previous two conditions (third column).

[0170] FIG. 11 shows Western Blot NP results for H1N1 infection with (TPCK+) or without (TPCK) trypsin in pancreatic islets. Influenza virus nucleoprotein was visualized as a band of 56 KDa.

[0171] FIGS. 12A, 12B, and 12C. Viral RNA detection by in situ hybridization in human pancreatic islet. Islets were infected with H1N1 and H3N2 adding 100 l of viral suspension containing viral dilution of 4.810.sup.3 pfu/well. Mock uninfected islets were left as a negative control. FIG. 12A: Two days after infection the presence of the virus RNA molecules was detected on cyto-embedded pancreatic islets upon addition of the Fast Red alkaline phosphatase substrate due to the formation of a coloured precipitate. Bound viral mRNA was then visualized using either a standard bright field or a fluorescent microscope (40X). Arrows: viral mRNA positive cells. FIGS. 12B and 12C: Five days after infection multiplex fluorescence-based in situ hybridization was performed and after disaggregation, islet cells were cytocentrifuged onto glass slides. Virus RNA, insulin, amylase and CK19 positive cells were assessed with a Carl Zeiss Axiovcrt 135TV fluorescence microscope. Quantification was performed using the IN Cell Investigator software. Each dot represents the percentage of positive cells quantified on one systematically random field. Results from two experiments performed are shown. Mann-Whitney U test was used for statistical analysis.

[0172] FIG. 13. Virus RNA and insulinlamylase/CK19 localisation. Figure shows multiplex histology data. Islets were infected with H1N1 and H3N2 adding 100 l of viral suspension containing viral dilution of 4.810.sup.3 pfu/well. Five days after infection multiplex fluorescence-based in situ hybridization was performed as described above. Left panels: the red signal corresponds to the presence of influenza virus RNA, the green signal corresponds to the presence of insulin, amylase or CK18 transcripts (63x). White arrow: double positive cells. Right panel: Virus RNA, insulin, amylase and CK19 positive cells were assessed with a Carl Zeiss Axiovert 1TV fluorescence microscope. Quantification was performed using the IN Cell Investigator software. Each dot represents the percentage of positive cells quantified on one systematically random field. Results from two experiments performed are shown.

[0173] FIGS. 14A and 14B. Islet survival and insulin secretion after infection with Human Influenza A Viruses. Islets were infected with H1N1 and H3N2 adding 100 l of viral suspension containing viral dilution of 4.810.sup.3 pfu/well. Mock uninfected islets were left as a negative control. The viabilities of pancreatic islets was evaluated 2, 5 and 7 days after infection. FIG. 14A shows light microscopy appearance of paraffin embedded islets 5 days after infection (20x) (upper). The viability (lower) was assessed using Live/Dead assay. Quantification was performed using the IN Cell Investigator software. Each dot represents the percentage of dead cells quantified on one random field. Results from two experiments (10 field each) are shown. FIG. 14B shows insulin secretion of isolated islets after culture for two days in the presence or in the absence of Human Influenza A Viruses. The figure shows insulin release after stimulation with glucose (2 to 20 mM) data are expressed as insulin secretion index calculated as the ratio between insulin concentration at the end of high glucose incubation and insulin concentration at the end of low glucose incubation, n=2.

[0174] FIGS. 15A and 15B. Cytokine/chemokine expression profile modification induced by Human Influenza A Viruses infection. Islets were infected with H1N1 and H3N2 adding 100 l of viral suspension containing two viral dilutions of 4.810.sup.3 or 4.810.sup.2 pfu/well. Mock uninfected islets were left as a negative control. Samples were collected every 48 hours from day of infection (t.sub.0) until day 10 (t.sub.10). The supernatant was collected and assayed for 50 cytokines.

[0175] FIG. 15A shows virus induced modification in islet cytokine/chemokine profile. Data are expressed as maximum fold increase for each factor detected during the culture respect mock infected islet (n=2). Dotted line: fivefold increase threshold. FIG. 15B shows IFN-gamma-inducible chemokines CXCL9/MIG, CXCL10/IP-10 concentration during ten day culture in the presence or in the absence of H1N1 and H3N2.

[0176] FIG. 16. Influenza virus M gene detection by RRT-PCR in pancreas and lungs of infected birds.

[0177] FIGS. 17A, 17B, 17C, 17D, and 17E. Immunohistochemistry for insulin. Pancreas, turkey. Representative islet structures before and after H3N7 at different time points.

[0178] FIG. 18. Receptor distribution profiles. Expression of alpha-2,3 and alpha-2,6-linked Sialic acid receptors on hCM, HPDE6 and MDCK cells. Shaded areas represent cells labelled with alpha-2,3 or alpha-2,6-specific lectins while unfilled areas represent unlabelled control cells. A minimum of 5,000 events were recorded per cell line.

[0179] FIGS. 19A, 19B, 19C, and 19D. Avian influenza virus replication kinetics in pancreatic cell lines. Replication kinetics of A/turkey/Italy/3675/1999 (H7N1) and A/turkey/Italy/2962/2003 (H7N3) in hCM and HPDE6 cells. hCM and HPDE6 cells were infected with each avian virus at an MOI=0.01 and at 24, 48 and 72 hours post-infection supernatants from three infected and one mock-infected control well were harvested for virus isolation and qRRT-PCR. (FIG. 19A) qRRT-PCR results of H7N1 in hCM and HPDE6. (FIG. 19B) qRRT-PCR results of H7N3 in hCM and HPDE6. (FIG. 19C) Virus isolation results of H7N1 in hCM and HPDE6. (FIG. 19D) Virus isolation results of H7N3 in hCM and HPDE6. All results represent means plus standard deviations of three independent experiments.

[0180] FIGS. 20A, 20B, 20C, and 20D. Immunofluorescence targeting the viral NP protein in pancreatic cell lines. (FIG. 20A) hCM negative control. (FIG. 20B) hCM at 24 hours post-infection (20X). (FIG. 20C) Nuclear staining of HPDE6 negative control (20X). The blue color corresponds to DAPT dye bound to DNA, while the red one is due to the Evans Blue contrast. (FIG. 20D) HPDE6 at 24 h post-infection (20X). The green signal corresponds to the presence of influenza virus NP protein derived from viral infection.

[0181] FIG. 21. Selected cytokines/chemokines, limits of detection and the coefficients of variability (intra Assay % CV and inter Assay % CV)

[0182] FIGS. 22 Group A and Group B. Viral shedding and viremia data.

MODES FOR CARRYING OUT THE INVENTION

[0183] Certain aspects of the present invention are described m greater detail in the non-limiting examples that follow. The examples are put forth so as to provide those of ordinary skill in the art with a disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0184] In this study the inventors explored the implications of influenza infection on pancreatic endocrine function in an animal model, and performed in vitro experiments aiming to establish the occurrence, extent and implications of influenza A virus infection in human cells of pancreatic origin. For the in vivo studies the inventors selected the turkey as a model because turkeys are highly susceptible to influenza infection and pancreatic damage is often observed as a post-mortem lesion. For the in vitro studies, the inventors selected A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2), as these viruses have circulated for extensive periods in humans, and existing epidemiological data would be suitable for a retrospective study. These strains were used to infect both established human pancreatic cell lines (including human insulinoma and pancreatic duct cell lines) and primary culture of human pancreatic islets.

[0185] In Vivo Experiments

[0186] Influenza A viruses originate from the wild bird reservoir and infect a variety of hosts including wild and domestic birds. These viruses are also able to infect a relevant number of mammals, in which they may become established. Among the latter there are swine, equids, mustelids, sea mammals, canids, felids and humans. IAV cause systemic or non-systemic infection depending on the strain involved. The systemic disease occurs mostly in avian species and is known as Highly Pathogenic Avian Influenza (HPAI). It is characterized by extensive viral replication in vital organs and death within a few days from the onset of clinical signs in the majority of infected animals. The non-systemic form, which is by far the most common, occurs in birds and in mammals and is characterised by mild respiratory and enteric signs. To differentiate it from HPAI, in birds it is known as low pathogenicity avian influenza (LPAI). This different clinical presentation resides in the fact that non-systemic influenza A viruses are able to replicate only in the presence of trypsin or trypsin-like enzymes and thus their replication is believed to be restricted to the respiratory and enteric tract.

[0187] IAV of avian origin have a tropism for the pancreas [5,88,89,90]. Necrotizing pancreatitis is a common finding in wild and domestic birds infected with HPAI [91,92,93,94] and the systemic nature of HP AI is in keeping with these findings. In contrast, it is difficult to explain pancreatic colonisation by LP AI viruses, which is a common finding in chickens and turkeys experiencing infection [95,96,97].

[0188] The aim of this study was to establish whether two natural non-systemic avian influenza viruses obtained from field outbreaks, without prior adaptation, could cause endocrine or exocrine pancreatic damage following experimental infection of young turkeys.

Animals

[0189] Sixty-eight female meat turkeys obtained at one day of age from a commercial farm were used in this study. Birds were housed in negative pressure, high efficiency particulate air (HEPA) filtered isolation cabinets for the duration of the experimental trial. Before carrying out the infection, animals were housed for 3 weeks to allow adaptation and growth, received feed and water ad libitum and were identified by means of wing tags.

[0190] Viruses

[0191] Two low pathogenicity avian influenza viruses (LPAI) isolated during epidemics in Italy were used for the experimental infection: A/turkey/Italy/3675/1999 (H7N1) and A/turkey/Italy/2962/2003 (H7N3). Both viruses had shown to cause pancreatic lesions in naturally infected birds. Stocks of avian influenza viruses were produced inoculating via the allantoic cavity 9-day-old embryonated specific pathogen free (SPF) chicken eggs. The allantoic fluid was harvested 48 hours post inoculation, aliquoted and stored at 80 C. until use. For viral titration, 100 l of 10-fold diluted viral suspension were inoculated in SPF embryonated chicken eggs and the median embryo infectious dose (EID.sub.50) was calculated according to the Reed and Muench formula.

Experimental Design

[0192] Animals were divided into three experimental groups [A (H7N1), B(H7N3) and K (control)]. Groups A and B, each constituted 24 animals, which were infected via the oro-nasal route with 0.1 ml of allantoic fluid containing 10.sup.6.83 EID.sub.50 of the A/turkey/Italy/3675/1999 (H7N1) virus and 10.sup.6.48 EID.sub.50 of the A/turkey/Italy/2962/2003 (H7N3) virus respectively. Group K, constituted animals, which received via the oro-nasal route 0.1 ml of negative allantoic fluid as negative control. All birds were observed twice daily for clinical signs. On days 0, 3, 6, 9, 13, 15, 20, 23, 27, 31, 34, 41 and 45 p.i. blood was collected from the brachial vein of all animals using heparinized syringes in order to determine glucose and lipase levels in plasma. On days 2 and 3 post infection (p.i.), tracheal swabs were collected to evaluate viral replication. On day 3 p.i., blood was also collected to determine the presence of viral RNA in the blood. On days 4 and 7 p.i., two birds from each infected group were humanely sacrificed and the pancreas and the lung were processed for the detection of viral RNA and for histopathology and immunohistochemistry. Similarly, on days 8 and 17 p.i., one subject from each experimental group was euthanized and the pancreas was collected for histological and immunohistochemical studies. For this purpose the inventors selected hyperglycaemic subjects that had also shown an increase in lipase levels.

Biochemical Analyses

[0193] Blood samples were collected in Gas Lyte 23 G pediatric syringes containing lyophilized lithium heparin as anticoagulant. At each sampling, 0.3 ml of blood was collected and refrigerated at 4 C. until processed. To obtain plasma, samples were immediately centrifuged at 1500g for 15 minutes at 4 C. To determine the levels of glucose and lipase in plasma, commercially available kits (Glucose HK and LIPC, Roche Diagnostics GmbH, Mannheim, Germany) were applied to the computerised system Cobas c501 (F. Hoffmann-La Roche Std, Basel, Switzerland). The Glucose HK test is based on an hexokinase enzymatic reaction. The linearity of the reaction is 0.11-41.6 mmoVL (2-750 mg/dL) and its analytic sensitivity is 0.11 mmol/L (2 mg/dL). The LIPC test is based on a colorimetric enzymatic reaction with a linearity of 3 a 300 U/L and an analytic sensitivity of 3 U/L.

Molecular Tests

[0194] Tracheal swabs, blood samples and organs (pancreas and lungs) were tested for viral RNA by means of RRT-PCR for the identification of the influenza virus Matrix (M) gene.

[0195] RNA extraction

[0196] Viral RNA was extracted from 100 l of blood using the commercial kit NucleoSpin RNA II (Macherey-Nagel) and from 50 l of phosphate buffered saline (PBS) containing tracheal swabs suspension using the Ambion MagMax-96 Al-ND Viral RNA Isolation Kit for the automatic extractor. 150 mg of homogenized lung and pancreas tissues were centrifuged and viral RNA was extracted from 100 l of clarified suspension using the NucleoSpin RNA II (Macherey-Nagel).

One Step RRT-PCR

[0197] The isolated RNA was amplified using the published primers and probes from reference 98, targeting the conserved Matrix (M) gene of type A influenza virus. 5 L of RNA were added to the reaction mixture composed by 300 nM of the forward and reverse primers (M25F and M124-R respectively), and 100 nM of the fluorescent label probe (M+64). The amplification reaction was performed in a final volume of 25 L using the commercial kit QuantiTect Multiplex RT-PCR kit (Qiagen, Hilden, Germany). The PCR reaction was performed using the following protocol: 20 minutes at 50 C. and 15 minutes at 95 C. followed by 40 cycles at 94 C. for 45 sec and 60 C. for 45 sec. Target RNA transcribed in vitro were obtained using the Mega Short Script 7 (high yield transcription kit, Ambion), according to the manifacturer' s instructions, quantified by NanoDrop 2000 (Thermo Scientific) and used to create a standard calibration curve for viral RNA quantification. To check the integrity of the isolated RNA, the -actin gene was also amplified using a set of primers in-house designed (primers sequences available upon request). The reaction mixture was composed by 300 nM of forward and reverse primer and IX of EvaGreen (Explera, Jesi, Italy). The amplification reaction was performed in a final volume of 254, using the commercial kit Superscript III (Invitrogen, Carlsbad, Calif.). The PCR reaction was performed using the following protocol: 30 minutes at 55 C. and 2 minutes at 94 C. followed by 45 cycles at 94 C. for 30 sec and 60 C. for 1 min.

Histology and Immunohistochemistry

[0198] Formalin-fixed, paraffin-embedded pancreas sections were cut (3 m thickness). Slides were stained with H&E (Histoserv, Inc., Germantown, Md.). Representative photos were taken with the SPOT ADVANCED software (Version 4.0.X, Diagnostic Instruments, Inc., Sterling Heights, Mich.). The reagents and methodology for Influenza THC were: Polyclonal Antibody Anti- type A Influenza Virus Nucleoprotein, Mouse-anti-Influenza A (NP subtype A, Clone EVS 238, European Veterinary Laboratory, 1:100 in PBS/2.5% BsA, for 1 hour at RT ; secondary antibody Goat-anti-mouse IgG2a HRP (Southern Biotech) 1/200 in PBS/2.5% BSA, for 1 hour at RT; Antigen retrieval was performed incubating the slides for 10 at 37 C. in trypsin (Kit Digest-all; Invitrogen); Endogenous peroxidase were blocked with 3% H.sub.2O.sub.2, for 10 at RT, before incubation with primary antibody slides a blocking step was performed with PBS/5% BSA for 20 at RT. DAB was applied as chromogen (Dakocytomation, ref. code K3468). IHC for insulin and glucagone: Polyclonal Guinea Pig Anti-Swine Insulin, 1:50 (A0564 Dako, Carpinteria, Calif.); Polyclonal Rabbit Anti-Glucagon, 1:200 (NCL-GLUC, Novocastra, Newcastle, UK) using as a detection system, the En Vision Ap (DAKO K1396, Carpinteria, Calif.) and nuclear fast Red (DAKO K1396) for the Influenza A staining; En Vision+System-HRP Labelled polymer Anti-Rabbit (K4002, Dako, Carpinteria, Calif.) and DAB (K3468, Dako, Carpinteria, Calif.) for Insulin and Glucagon staining.

In Vitro Assays

[0199] The aims of these experiments were to establish whether human influenza viruses can grow on human primary and established cell lines derived from the human pancreas, and the effect of their replication on primary cells.

Cell Lines

[0200] Maclin Darby Canine Kidney (MDCK) cells were maintained in Alpha's Modified Eagle Medium (AMEM, Sigma) supplemented with 10% Foetal Bovine Serum (FBS), 1% 200 mM L-glutamine and a 1% penicillin/streptomycin/nystatin (pen-strep-nys) solution. The human insulinoma cell line CM [99] and immortalized human ductal epithelial cell line HPDE6 [100] were maintained in RPMI (Gibco) supplemented with 1% L-glutamine, 1% antibiotics and FBS (5% and 10%, respectively). MDCKs and HPDE6 were passaged twice weekly at a subcultivation ratio of 1:10 and 1:4, while CM were split three times per week at a ratio of 1 :4. All cells were maintained in a humidified incubator at 37C with 5% CO.sub.2.

Primary Cells

[0201] Pancreatic islets were isolated and purified at San Raffaele Scientific Institute from pancreases of multiorgan donors according to Ricordi's method. Islet preparations with purity >80%8% (meanSD, n=6) not suitable for transplantation, were used after approval by the local ethical committee. Cells were seeded in 24 well plates and 25 cm2 flasks at 150 islets/ml and maintained in final wash culture medium (Mediatech, Inc., Manassas, Va.) medium at 37 C. with 5% CO.sub.2.

Sialic Acid Receptor Characterization on CM and HPDE6 Cells

[0202] The presence of alpha-2,3 and alpha-2,6-linked sialic acid residues was determined via flow cytometry. Following trypsinization, 110.sup.6 cells washed twice with PBS-10 mM HEPES (PBS-HEPES), for 5 minutes at 1200 RPM, and then treated with an Avidin/Biotin blocking kit (Vector Laboratories, USA) as per manufacturer's instructions, with cells incubated for 15 minutes with 100 l of each solution. Alpha-2,3 and alpha-2,6 sialic acid linkages, respectively, were detected by incubating cells for 30 minutes with 100 l of biotinylated Maackia amurensis lectin II (Vector Laboratories) (5 g/ml) followed by 100 l of PE-Streptavidin (BD Biosciences) (10 g/ml) for 30 minutes at 4C in the dark, or with 100 l of Fluorescein conjugated Sambucus nigra lectin (Vector Laboratories) (5 g/ml). Cells were washed twice with PBS-HEPES between all blocking and staining steps and resuspended in PBS with 1% fonnalin prior to analysis. To confirm specificity of lectins, cells were pre-treated with 1 U per mL of neuraminidase from Clostridium peifringens (Sigma) for one hour prior to the avidin/biotin block. Samples were analyzed on a BD Facscalibur or the BD LSR II (BD Biosciences) and a minimum of 5,000 events were recorded.

Viruses and Viral Titration

[0203] Stocks of A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2), referred as H1N1 and H3N2 respectively, were produced in cell culture or in embryonated chicken eggs. Viruses were titrated by standard plaque assay.

[0204] To propagate IAV, monolayer cultured MDCK cells were washed twice with PBS and infected with A/NewCaledonia/20/99 (H1N1) or A/Wisconsin/67/05 (H3N2) at an MOI of 0.001. After virus adsorption for 1 h at 35 C., the cells were washed twice and incubated at 35 C. with DMEM without serum supplemented with TPCK-treated trypsin (1 g/ml, Worthington Biomedial Corporation, Lakewood, N.J., USA). Supernatants were recovered forty-eight hours post-infection. Low Pathogenicity avian influenza viruses (LPAI) H7N1 A/turkey/Italy/3675/1999 and H7N3 A/turkey/Italy/2962/2003 isolated during epidemics in Italy were grown in 9-day-old embryonated specific pathogen free (SPF) chicken eggs as described in section 2.1.2. For viral titration, plaque assays were performed as previously described [101]. Briefly, MDCK monolayer cells, plated in 24-well plates at 2.510.sup.5 cells/well, were washed twice with DMEM without serum, and serial dilutions of virus were adsorbed onto cells for 1 hour. Cells were covered with MEM 2XAvicel (FMC Biopolymer, Philadelphia, Pa., USA) mix supplemented with TPCK-treated trypsin (1 g/ml). Crystal violet staining was performed 48 hours post-infection and visible plaques were counted.

Virus Replication Kinetics in Pancreatic Cell Lines

[0205] Semi-confluent monolayers of HPDE6 and CM cells seeded on 24-well plates were washed twice with PBS and then infected at an MOI of 0.001 using 200 l of inoculum per well. Inoculum was removed after one hour of absorption and replaced with 1 ml of serum-free media containing 0.05 g/l TPCK-Trypsin (Sigma). At 1, 24, 48 and 72 hours post-infection supernatants from three infected wells and one control well were harvested, and viral titres were determined by virus isolation using the 50% tissue culture infectious dose (TCID.sub.50) assay as well as by Real Time RT-PCR detection of the Matrix gene. All replication kinetics experiments were repeated three times.

TCID.SUB.50..

[0206] Confluent monolayers of MDCK cells seeded onto 96-well plates were washed twice in serum-free medium and inoculated with 50 l of 10-fold serially diluted samples in serum free MEM. After one hour of absorption an additional 50 l of serum-free media containing 2 g/ml TPCK-Trypsin was added to each well. CPE scores were determined after three days of incubation at 37 C. by visual examination of infected wells on a light microscope. The TCID.sub.50 value was determined using the method of Reed and Muench.

Growth Assay in Pancreatic Islets

[0207] Islets were infected with H1N1 and H3N2 influenza viruses adding 4.810.sup.2 or 4.810.sup.3 pfu/well. Viral growth was performed with and without the addition of TPCK trypsin (SIGMA) (1 g/ml). Uninfected islets were left as a negative control. Samples were collected every 48 hours from day of infection (t.sub.0) until day 10 (t.sub.5). Each sample was centrifuged at 150 g for 5 minutes. The supernatant was collected and stored at 80 C. for quantitative Real Time PCR, virus titration and cytokine expression profile. The pellet was washed twice with PBS, stored at 80 C. and subsequently processed for Real Time PCR, Western Blot and virus titration in MDCK cells, see above). All pellets and supernatants were tested for Real Time PCR in triplicate.

Detection of Viral RNA (Rom Pancreatic Tissue

[0208] The total RNAs from pancreatic islet pellets and supernatants were isolated using the commercial kit NucleoSpin RNA II (Macherey-Nagel) according to the manifacturer' s instructions. RNAs were eluted in 60 l of elution buffer and tested using One step RRT-PCR for influenza Matrix gene (see below) to evaluate the viral growth.

[0209] A quadratic regression model (Ct=.sub.0+.sub.1TPCK-trypsin+.sub.2time+.sub.3time.sup.2+.sub.4time.Math.TPCK-trypsin .sub.5time.sup.2 TPCK-trypsin) for each viruses and specimen was used to analyse the trend of Ct value over time. The influence of TPCK presence and the interaction between its presence and time point was evaluated. The regression model took into account the influence of the intra-group correlation among repeated measurements for each observed time in the confidence intervals (CIs) calculation. A residuals post-estimation analysis was performed to verify the validity of the model.

One Step RRT-PCR

[0210] Quantitative Real Time PCR, targeting the conserved Matrix (M) gene of type A influenza virus, was applied according to the protocol described in section 2.1.5 above. To check the integrity of the isolated RNA, the -actin gene was also amplified using primers and probe previously described [102]. The reaction mixture was composed by 400 nM of forward and reverse primer (Primer-beta act intronic and Primer-beta act reverse respectively) and 200 nM of the fluorescent label probe (5-Cy5 3-BHQ1). The amplification reaction was performed in a final volume of 25 L using the commercial kit QuantiTect Multiplex RT-PCR kit (Qiagen, Hilden, Germany). The PCR reaction was using the following protocol: 20 minutes at 50 C. and 15 minutes at 95 C. followed by 45 cycles at 94 C. for 45 C. and 55 C. for 45 sec.

Western Blot Analysis

[0211] Cellular pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8; 1.0% SDS; 350 mM NaCl; 0.25% Triton-X; proteases inhibitor cocktail) then mixed and incubated on ice for 30 minutes. The suspension was sonicated three times for 5 minutes each and then centrifuged at maximum speed for 10 minutes. Bradford test was performed in order calculate the total protein concentration for each sample. Based on this calculation the same amount of protein/sample was treated in dissociation buffer (50 mM Tris-Cl, pH 6.8; 5% -mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) for 5 minutes at 96 C. and then electrophoresed in 12% polyacrilamide gels using running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). Following SDS-PAGE the proteins were transferred from the gel onto immuno-blot PVD membranes (Bio-Rad) by electroblotting with transfer buffer (39 mM glycine, 48mM Tris base, 0.037% SDS, 20% methanol). Membranes were washed with PBS and then incubated overnight at 4 C. in 5% dried milk in PBS. After washing with PBS membranes were incubated for 1 h at room temperature under constant shaking in PBS containing 0.05% Tween-20 (SIGMA), 5% blotting grade blocker non-fat dry milk (Bio-Rad) and mouse monoclonal Influenza A virus Nucleoprotein antibody (Abcam). Beta Actin antibody (Abcam) was used as loading control. After incubation with the primary antibody, membranes were exposed for 1 h to horseradish peroxidise-(HRP) rabbit polyclonal secondary antibody to mouse TgG (Abcam), followed by visualization of positive bands by ECL using Hyperfilm ECL (Amersham Biosciences).

Visualisation of Viral Growth in Pancreatic Cell Lines

[0212] HPDE6 and hCM cells were grown in slides to 80% confluence and infected with either H1N1 or H3N2 viruses at an M.O.I. of 0.1 with 0.05 mg/ml of TPCK. Cells were fixed and permeabilized at 0, 24, 48 and 72 h p.i. with chilled acetone (80%). After blocking with PBS containing 1% BSA, the cells were incubated for 1 h at 37 C. in a humidified chamber with mouse monoclonal to influenza A virus nucleoproteinFITC conjugated (Abcam) in PBS containing 1% BSA and 0.2% Evan's Blue. The staining solution was decanted and the cells were washed three times. Nuclei of negative control cells were stained with DAPI (SIGMA), then washed with PBS and observed under UV light.

In Situ Visualisation of Viral RNA in Pancreatic Islets

[0213] To visualize viral RNA localized within cells, purified human pancreatic islets were harvested at 2, 5 and 7 days post infection. Islets were then incubated for 24 h in methanol-free 10% formalin, deposited at the bottom of flat-bottomed tubes, embedded in agar to immobilize them, dehydrated, and finally embedded in paraffin. Islet samples were sectioned at 4 mm. For co-ocalization experiments, islets were harvested 5 days post infection, enzymatically digested into single cells with a trypsin-like enzyme (12605-01, TrypLE Express, Invitrogen, Carlsband, California) and cytocentrifuged onto glass slides. In situ hybridization was performed using the Quantigene ViewRNA technique, based on multiple oligonucleotide probes and branched DNA signal amplification technology, according to the manufacturer instructions (Affymetrix, Santa Clara, Calif., USA). The probe set used was designed to hybridize the H1N1/A/New Caledonia/20/99 virus (GenBank sequence: DQ508858.1). Due to sequence homology in the region covered by the probes, the same set recognized also the H3N2 virus RNA as confirmed in pilot experiments. To identify cell types within islets the following Quantigene probes were used: insulin for beta cells (INS gene, NCBI Reference Sequence: NM_000207); alpha-amylase 1 for exocrine cells (AMY1A gene, NCBI Reference Sequence:NM_004038); cytokeratin 19 for duct cells (KRT19 gene, NCBI Reference Sequence: NM_002276). Quantification of cells positive for each probe was performed within 8 randomly chosen fields using the IN Cell Investigator software (GE Healthcare UK Ltd).

Determination of Insulin Secretion in Infected Islets

[0214] Aliquots of 100 islet equivalents (uninfected or infected with H1N1/A/New Caledonia/20/99 and H3N2/A/Wisconsin/67/05) per column were loaded onto Sephadex G-10 columns with media at low glucose concentration (2mM) and preincubated at 37 C. for 1 hour. After preincubation, islet were exposed to sequential 1 hr incubations at low (2 mM) and high (20 mM) glucose concentration. Supernatants were collected with protease inhibitors cocktail (Roche Biochemicals, Indianapolis, Ind.) and stored at 80 C. at the end of each incubation. Insulin content was determined with an insulin enzyme-linked immunoassay kit (Mercodia AB, Uppsala, Sweden) following manufacter's instruction. Insulin secretion index were calculated as the ratio between insulin concentration at the end of high glucose incubation and insulin concentration at the end of low glucose incubation

Cytokine Expression Profile

[0215] The capability of H1N1 and H3N2 viruses to induce cytokine expression in human pancreatic islets was measured using multiplex bead-based assays based on xMAP technology (Bio-Plex; Biorad Laboratories, Hercules, Calif., USA). The parallel wells of pancreatic were infected with viruses or were mock infected. The culture media supernatant was collected before and 2, 4, 6, 8, 10 days post infection and assayed for 48 cytokines. Selected cytokines, limits of detection and the coefficients of variability (intra Assay % CV and inter Assay % CV) of the cytokine/chemokine are shown in FIG. 21.

Evaluation of Cell Death Following Infection (Live/Dead Assay)

[0216] The viability of islet cells after infection was measured using the live/dead cell assay kit (L-3224, Molecular Probes, Inc., Leiden, The Netherlands). The assay is based on the simultaneous determination of live and dead cells with two fluorescent probes. Live cells are stained green by calcein due to their esterase activity, and nuclei of dead cells are stained red by ethidium homodimer-1. Islets harvested after five days of culture were further enzymatically digested into single cells with trypsin-like enzyme (12605-01, TrypLE Express, Invitrogen, Carlsband, Calif.). According to manufacturer's instructions single cells were incubated with the labeling solution for 30 min at room temperature in the dark, cytocentrifuged onto glass slides, and assessed with a Carl Zeiss Axiovert 135TV fluorescence microscope. Analysis of dead cells were performed on cytospin preparations using the IN Cell Investigator software (GE Healthcare UK Ltd). Positive cells in each category were quantified with 10 systematically random fields.

Statistical Analysis

[0217] Data were generally expressed as meanstandard deviation or median (Min-Max). Differences between parameters were evaluated using Student's T test when parameters were normally distributed, Mann Whitney U test when parameters were not normally distributed. Kaplan-Meier and/or Cox regression Analysis was used to analyze incidence of event during the time. A p value of less than 0.05 was considered an indicator of statistical significance. Analysis of data was done using the SPSS statistical package for Windows (SPSS Inc., Chicago, Ill., USA).

RESULTS

In Vivo Experiment

Clinical Disease

[0218] Turkeys from both H7N1 [A] and H7N3 [B] challenged groups showed clinical signs typical of LPAI infection, such as conjunctivitis, sinusitis, diarrhoea, ruffled feathers and depression on day 2 p.i. Mild symptoms regressed by day 20 p.i. Only two subjects from group A showed sinusitis until day 30 p.i. Mortality rate was low in both groups: one subject of group A died on day 8 p.i. and one subject of group B died on day 19 p.i.

Detection of Viral RNA

[0219] Viral RNA was detected from the tracheal swabs collected from 17/20 subjects infected with H7N1 and 19/20 subjects infected with H7N3 on day 2 and all animals on day 3 p.i. Viral RNA was also detected from the blood of two subjects of group A H7N1 and four subjects of group B H7N3 on day 3 p.i., (FIG. 22 Group A and Group B) and from the pancreas and lungs collected on days 4 and 7 p.i. (FIG. 16). No viral RNA was detected from the uninfected controls.

Biochemical Analyses

[0220] In blood samples collected intra-vitam to reveal metabolic alterations, a significant increase in plasmatic lipase levels (10 to 100 times the values of the control animals) was evident in H7N1 (12/20) and H7N3 (10/20) challenged turkeys between day 3 and 9 p.i. (FIGS. 2A and 2B) while none of uninfected controls showed modification of lipase levels (20/20; p<0.001, Pearson Chi-Square). A clear trend between the presence of viral RNA in blood at day 3 and the increase in lipase was evident in infected animals (Hazard Ratio 2.51 with 95% confidence interval 0.92 to 6.81; p 0.07). Lipase levels within the normal range were rapidly re-established in all cases, reason for which on day 23 p.i., it was decided to no longer evaluate this parameter on day 23 (FIGS. 1A, 1B, and 1C). After day 9 p.i. 5 animals of group A and 5 animals of group B developed hyperglycaemia (FIGS. 2A and 2B). Of these, two subjects maintained the hyperglycaemic status throughout the entire experiment while in all the other animals the levels of blood glucose returned similar to those of controls (FIGS. 1A, 1B, and 1C). A clear association between the increase in lipase between day 3 and 9 p.i. and the development of hyperglycaemia after day 9 p.i. was evident. In fact, hyperglycaemia was present only in the subjects who developed high lipase values post infection while never appeared in subject with normal lipase level (10/22 and 0/18 respectively, p=0.001) with a median time between hyperlipasemia and hyperglycaemia developments of 4.5 days (min-max: 3-7).

Histopathology and Immunohistochemistry

[0221] None of the control turkeys showed significant histological changes or positive immunohistological reactions against ATV (FIG. 3). In all infected birds, histopathologic lesions were evident, although markedly different in samples collected at different timings post infection. At early stages (day 4-8 p.i.), an acute pancreatitis with necrotic acinar cell, massive inflammatory infiltration composed of macrophages, heterophils, lymphocytes and plasmacells dominates over areas of healthy/uninvolved/spared tissue (FIG. 4). From day 8 p.i., these necrotic inflammatory lesions were gradually replaced by ductules and lymphocytic infiltration with mild degree of fibroplasia. At later stages (day 17 p.i) extensive fibrosis, with lymphoid nodules replaced pancreatic parenchyma and disruption of the normal architecture of the organ were evident (FIG. 5). Variable numbers of necrotic acinar cells were observed during all the experimental period. Obstructive ductal lesions were not seen in any case and stage.

[0222] By immunohistological staining, degenerating and necrotic acinar cells showed specific reaction to virus nucleoprotein antigen antibody during the experimental period (FIG. 6). Some of the vascular endothelial cells also showed positive reaction, as well as occasional ductal epithelial cells. In uninfected controls the insulin positive tissues of the pancreas were scattered singly or in small groups of islets of various shapes and sizes in the intersititium of the exocrine part (FIG. 17A). At day 8 p.i. the normal structure of islets was partially destroyed and the number of islet cells was reduced. Remaining islets were smaller and distorted, with irregular outlines or dilated intra-islet capillaries; the number of cells staining for insulin was also reduced: these cells presented enlarged cytoplasm and sometimes appeared to have granular degeneration and even necrosis. Fibrous bands appeared inside the islet with islet fragmentation and dislocation of small and large clusters of endocrine cells (FIG. 17B). At day 17 p.i. separated large clusters of endocrine insulin positive cells were evident embedded in or close to the extensive fibrosis that replaced the acinar component (FIG. 17C). Beyond day 17 p.i. groups of very large (>200 m in diameter), usually irregular, islet like areas of mainly insulin immunoreactivity were clearly present scattered in extensive acinar fibrosis (FIGS. 17D and 17E).

In Vitro Experiment

[0223] Susceptibility of Human pancreatic cell lines to Human Influenza A Viruses

[0224] The susceptibility of endocrine (hCM, insulinoma) and ductal (HPDE6) cell lines to H1N1/A/New Caledonia/20/99 and H3N2/A/Wisconsin/67/05 infections were investigated.

Receptor Distribution

[0225] Lectin staining of both the hCM and HPDE6 cell lines revealed high levels of alpha-2,6 sialic acid-linked sialic acids molecules (required by human-tropic viruses) as well as alpha-2,3 linked residues (used by avian-tropic viruses). The mean peak intensities of hCM incubated with Maackia amurensis lectin II (alpha-2,3 specific) and Sambucus nigra lectin (alpha-2,6-specific), were nearly identical, at approximately 2.610.sup.4 for both receptors. HPDE6 also had high level expression of both receptor types, with 3.710.sup.4 for SNA and 1.610.sup.4 for MAA. MDCK cells were also included as a positive control line for both receptor types as these cells are widely used for the isolation of human and avian origin viruses. FACS analysis showed MDCKs expressed similar levels of alpha-2,3 receptors to the HPDE6, with mean peak intensity ncar 1.810.sup.4, while alpha-2,6 expression was equal to that of hCM, with a mean fluorescence at 2.510.sup.4. Therefore, both pancreatic cell lines can be said to express sialic acid receptors in levels comparable to MDCKs, and in the case of hCM expression of the human-virus receptors was even higher (FIG. 18). Pre-treatment of all cells with 1 U/ml of NA from Clostridium peifringens resulted in decreased fluorescence for both lectin types, confirming specificity (data not shown).

Virus Replication Kinetics in Pancreatic Cell Lines

[0226] hCM and HPDE6 cells were infected with H1N1 and H3N2 viruses at a MOI=0.001. Visual examination of the infected cells by light microscopy revealed no cytopathic effect at any time point post-infection on hCM or HPDE6. TCID50 results revealed a continued increase in viral titres in HPDE6 over the 72 hour course, though the H1N1 viral titres were only slightly higher at 72 hours compared to 48 hours post-infection. In contrast, viral titres reached in hCM cells remained quite similar from 48 to 72 hours post-infection in the case of both H1N1 and H3N2 isolates (FIGS. 17A and 17C). An examination of viral RNA replication by qRRT-PCR showed a continued increase in viral replication up to 72 hours post-infection in both cell lines and for both viruses tested (FIGS. 17B and 17D).

[0227] Despite the higher M.O.I used to perform the infections (M.O.I=0.01) avian influenza virus showed lower levels of replication in both pancreatic cell lines compared to the human viruses (FIGS. 19A, 19B, 19C, and 19D), with a trend characterized by steady levels of virus RNA up to 48 hours p.i. and a decrease for both cell lines at 72 hours p.i. Based on the RRT-PCR results, hCM appeared to be more sensitive to avian viruses since the total amount of M gene RNA on average resulted 2 logs higher than HPDE6 (FIG. 19A and 19B). This was confirmed also by TCID50 results (FIG. 19C and 19D), in which both viruses reached higher titres in hCM. In the latter, however the H7N1 strain exhibited a higher replication efficacy in compared to H7N3. This result is not reflected in the RRT-PCR results for which comparable amounts of viral RNA were detected for both viruses. No significant differences in the viral replication between the two avian viruses were observed in HPDE6.

Western Blot Analysis for Detection of Virus Nucleoprotein

[0228] Results of H1N1 and H3N2 influenza virus nucleoprotein in hCM and HPDE6 cell lines are reported in FIGS. 8A, 8B, 8E, and 8F. No differences, depending either on the viral strain or on the cell type, were shown in the trend of NP expression. As expected influenza virus nucleoprotein was not observed at to (before infection), while it was detected at 24 (t.sub.24), 48 (t.sub.48) and 72 (t.sub.72) hours post-infection for both viruses in hCM as well as in HPDE6. Comparing the bands obtained from samples at t.sub.24 to those obtained at t.sub.48 and t.sub.72 an increase in the NP expression was observable. On the other hand the amount of beta actin, used as loading control, was at the same levels in all the samples tested (FIGS. 8C, 8D, 8G, and 8H).

Immunofluorescence Targeting the NP Protein

[0229] Human influenza virus replication was also detected by a fluorescent signal derived from FITC conjugate in hCM at 24 h post-infection (FIGS. 20A and 20B) for both viruses tested and increased over time at 48 and 72 hours post-infection. No differences were observed between the viral stains tested. The fluorescence signal for both viruses observed at 24 h post-infection in HPDE6 cells (FIGS. 20C and 20D). Also, in this case the number of cells marked continued to increase at 48 and 72 h post-infection, demonstrating the enhancement of the nucleoprotein expression over time (data not shown).

Susceptibility of Human Pancreatic Islet to Human Influenza A Viruses

[0230] The regression model indicated that the Ct values for both viruses, in presence or in absence of TPCK-trypsin, tested in both in pellets or in supernatant specimens, decreased significantly over time (p<0.05) (FIGS. 10A, 10B, 10C, and 10D). The statistical analysis showed that the virus titer increased over time independently of the virus subtype and type of sample (pellet or supernatant). Interestingly, only for H1N1 pellets and supernatant samples Ct values for the viruses grown with TPCK-trypsin decreased significantly more than those obtained without the exogenous proteases (p<0.01) (FIGS. 10A and 10C). TPCK-trypsin seemed to enhance H3N2 virus growth but the difference did not reach statistical significance (p>0.10) (FIG. 11). The residuals post-estimation analysis indicates that the model used was appropriate (data not shown).

[0231] In situ hybridization was performed to visualize viral RNA localized within islet cells. The results clearly demonstrate the presence of viral RNssA both after H1N1 and H3N2 infection (FIG. 12A). Since human islet primary cultures contain both endocrine and exocrine cells a fluorescence-based multiplex in situ hybridization strategy was applied to determine which and how many cells were infected in the islets. For this purpose distinctly labelled probes were combined to analyze viral RNA and insulin, amylase or cytokeratin 19 transcripts simultaneously and, after hybridization, human islets were disaggregated and cells positivity quantified. Five days after infection 0%, 10.8% and 4.3% of total cells resulted positive for viral RNA in mock, H1N1 and H3N2 infected islets, respectively (p<0.001) (FIG. 12B). Of the H1N1 positive cells 4927% stained positive for insulin, 2616% for amylase, 1.62.4% for CK19 and 2521% were negative for tested transcripts. Of the H3N2 positive cells 4023% stained positive for insulin 2020% for amylase, 2.3+5% for CK19 and 4145% were negative for tested transcripts (FIG. 12C). On the other hand, of the insulin positive cells 1410% and 88% were positive for viral RNA 5 days after H1N1 and H3N2 infection respectively (p=0.023). Of the amylase positive cell 279% and 96% were positive for viral RNA after H1N1 and H3N2 infection, respectively (p<0.001). Of the CK19 positive cell 34% and 1.33% were positive for viral RNA after H1N1 and H3N2 infection, respectively (p=0.36) (FIG. 13).

Modulation of Survival, Insulin Secretion and Innate Immunity in Human Pancreatic Islets Infected with Hwnan Influenza A Viruses In Vitro.

[0232] Visual examination of the infected islets by light microscopy and Live/Dead assay revealed no significant cytopathic effect at any time point post-infection (day 0-7). Five days after infection, uninfected cells showed an overall mortality of 3.26%, H3N2 of 5.21% and H1N1 of 7.38% (p=ns vs mock infected cell) (FIGS. 14A and 14B). Moreover exposure of islets to both H1N1 and H3N2 did not affect their ability to respond to high glucose, as tested in a static perfusion system (FIGS. 14A and 14B).

[0233] The capability of H1N1 and H3N2 to induce cytokine/chemokines expression m human pancreatic islet was measured using multiplex bead-based assays based on xMAP technology. The parallel wells of human islets (150 islets/well) were infected with HINI and H3N2 at 102 103 pfu/well, or they were mock infected. The culture media supernatant was collected at five time points (0, 4, 6, 8, 10 days) post infection, and assayed for 50 cytokines. With the exception of three (1L-1b, 1L-5, 1L-7) all the cytokines showed detectable expression. In mock infected the highest concentrations were detected for CCL2/MCP1 (max 25,558 pg/ml, day 4), ICAM-1 (max 14,063, day 1 0), CXCL8/IL-8 (max 11,6 pg/ml, day 1 0); IL-6 (8,452 pg/ml, day 4), CXCL1/GRO- (max 8,581 pg/ml, day 4), VCAM-1 (max 5,566 pg/ml, day 6) VEGF (max 3,225 pg/ml, day 10), SCGF-b (max 1,427 pg/ml, day 6), HGF (max 1,195 pg/ml, day 6). MIF (max 806 pg/ml, day 6), G-CSF (max 794 pg/ml day 6), CXCL9/MIG (max 448 pg/ml, day 6) GM-CSF (max 280 pg/ml, day 4), IL-2Ra (max 230 pg/ml, day 6), IL-12p40 (max 215 pg/ml, day 6), M-CSF (max 212 pg/ml, day 10), LIF (max 185 pg/ml, day 6), CXCL4/SDF1 (max 121 pg/ml, day6) showed lower but consistent expression. CXCL10/IP-10, PDGF-BB, IL-1Ra, IL-12p70, CCL11/Eotaxin, FGFb, CCLS/RANTES, CCL4/MIP-1, CCL7/MCP-3, IL-3, IL-16, SCF, TRAIL, INFa2, INFg, CCL27/CTAK showed low but consistent expression (max between 10 to 100 pg/ml). Very low (max <10 pg/ml) but detectable expression was present for IL-2, IL-4, IL-9, IL10, IL-13, IL-15, CCL3/MIP-, TNF-, IL-17, IL-18, IL1, -NGF, TNF-. Two inflammatory cytokines (IL-6, TNF) and six inflammatory chemokines (CXCL8/IL-8, CXCL1/GRO-, CXCL9/MIG, CXCL10/IP-10, CCLS/RANTES, CCL4/MIP-1) showed over fivefold increase in influenza viruses-infected cell supernatants compared to mock-infected controls (FIG. 15A). Between these the INF- inducible chemokines CXCL9/MIG, CXCL10/IP-10 showed the strongest response to H1N1 or H3N2 infection (over one hundred fold increase). Both peaked 6-8 days post infection and showed a stronger response to higher dose of viruses (FIG. 15B).

Summary of Results

[0234] The objective of this work was to assess IAV replication in pancreatic cells and to evaluate its consequence both at cellular level in vitro and at tissue level in vivo. These studies indicate, for the first time, that human influenza A viruses are able to grow in human pancreatic primary cells and cell lines. The addition of exogenous trypsin appears to enhance viral replication, but is surprisingly not essential for viral replication in human pancreatic primary cells and cell lines. The inventors' in vivo results confirmed these findings, where two non-systemic strains of IAVs were able to colonise the pancreas of experimentally infected poults and with metabolic consequences that reflect endocrine and exocrine damage.

[0235] The colonisation of the pancreas by IAV has been reported following a number of natural and experimental infections of animals, primarily in birds undergoing both systemic and nonsystemic infection (see references above). However, there is no direct evidence of infection of the pancreas in humans. Here, the inventors have demonstrated for the first time that two non-systemic avian influenza viruses cause severe pancreatitis resulting in a dismetabolic condition comparable with diabetes as it occurs in birds. Literature is available on the clinical implications of endocrine and exocrine dysfunctions of the pancreas in birds, including poultry. Regarding endocrine function, several studies indicate that with a total pancreatectomy birds suffer severe hypoglycaemic crisis leading to death [103]. If a residual portion of the pancreas as small as 1% of the pancreatic mass is left in situ, a transient (or reversible) hyperglycaemic condition is observed in granivorous birds, in which, normal glycemia is re-established within a couple of weeks [104,105]. This indicates that the pancreatic tissue of birds has significant compensatory potential and is also influenced by the fact that there is evidence towards the presence of some endocrine tissue able to secrete insulin outside the pancreas [106]. Insulin is the dominant hormone in the well-fed bird, while glucagon is the dominant hormone in the fasting bird. In this experiment, which was carried out with food ad libitum, damage of the endocrine component of the pancreas, would likely manifest itself with hyperglycemia.

[0236] Regarding exocrine function, pancreatitis in birds is characterised by malaise, reluctance to feed, enteritis and depression. Intra-vitam investigations are based on increased haematic lipase concentration [105]. In this study pancreatitis was evaluated by measuring the lipase concentration in the blood stream, and by histopathologic examination of pancreas collected at different time points. As it occurs in mammals, pancreatic damage determined a rapid increase of the haematic lipase levels which was transient and the values returned to normal by day 15 p.i. Interestingly, the birds which had shown the increased lipase levels in the blood and thus supposedly the most severe pancreatic damage, exhibited in the subsequent days high blood glucose levels, which only in a few cases persisted until the termination of the experiment. This is in-keeping with the clinical and metabolic presentation of diabetes in birds. The histological investigations clearly indicate viral replication in the exocrine portion of the pancreas, resulting in fibrosis and disruption of the organ's architecture. While it is clear that both isolates under study replicated extensively in the acinar component of the pancreas, the inventors were unable to determine whether viral replication also occurred in the islets. Based on these results, the inventors suggest that influenza virus infection caused severe acute pancreatitis which has impaired both the endocrine and exocrine functions.

[0237] Current knowledge on influenza replication indicate that influenza viruses which do not exhibit a multibasic cleavage site of the HA protein do not become systemic. However, in the in vivo experiments the virus reached the pancreas, and the inventors have surprisingly detected viral RNA on day 3 post infection from the blood in 2/20 (Group A- H7N1) and 4/20 (Group B-H7N3) infected turkeys. The inventors postulate that, following replication in target organs such as the lung and the gut, in some individuals, a small amount of virus reaches the bloodstream and thus the pancreas. Although the detected Ct values detected indicate low levels of viral RNA, this often resulted in the development of pancreatitis (detected in vivo by hyperlipasemia). This in turn, in the experimental model has resulted in an hyperglycaemic condition, consistent with the presentation of diabetes in granivorous birds.

[0238] The results of the in vitro experiments show that all IAVs tested, both of avian (H1N1 and H7N3) and of human origin (HINI Caledonia/20/99 and H3N2 A/Wisconsin/67/2005) are able to grow in established pancreatic cell lines and in pancreatic islets. Viral replication occurs both in cells of endocrine and exocrine origin. These investigations also show that both alpha-2-3 and alpha-2-6 receptors are present in pancreatic cells, indicating that both human and avian influenza viruses could find suitable receptors in this organ. The human viruses used in this study did not induce a significant mortality of islet cells, and insulin secretion did not appear to be affected by infection in this system. On the other hand, it was clear from the cytokine expression profile that IAV infection is able to induce a strong pro-inflammatory program in human pancreatic islets. The INF-gamma-inducible chemokines MIG/CXCL9/and IP-10/CXCL10 showed the highest increase after infection. Also huge amounts of RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, IL8/CXCL8, TNFa and IL-6 were released. Of interest, many of these factors were described as key mediators in the pathogenesis of type 1 diabetes [107].

[0239] Recently 1P10/CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients whereas RANTES/CCL5 and MIG/CXCL9 proteins were present at lower levels in the islets of both species [108]. The chemokine IP-10/CXCL10 attracts monocytes, T lymphocytes and NK cells, and islet-specific expression of CXCL10 in a mouse model of autoimmune diabetes caused by viruses [rat insulin promotor (RIP)-LMCV] accelerates autoimmunity by enhancing the migration of antigenspecific lymphocytes [109]. This is in keeping with bother findings in which neutralization of IP-10/CXCL10 [110] or its receptor (CXCR3) [111] prevents autoimmune disease in the same mouse model (RIP-LCMV). Studies in NOD mice have demonstrated elevated expression of IP-10/CXCL10, mRNA and/or protein in pancreatic islets during the prediabetic stage [112]. Increased levels of MIP1b/CCL4 and IP-10/CXCL10 are present in the serum of patients who have recently been diagnosed as having type 1 diabetes [113,114].

[0240] The inventors propose that, if influenza virus finds its way to the pancreas, either through viraemia, as detected in human patients [115,116, 117], or through reflux from the gut through the pancreatic duct, the virus would find a permissive environment. Here, the virus would encounter appropriate cell receptors and susceptible cells belonging to both the endocrine and exocrine component of the organ. Viral replication would result in cell damage due to the activation of a cytokine storm similar to the one associated with various conditions linked to diabetes. Thus the inventors believe that influenza infections may lead to pancreatic damage resulting in acute pancreatitis and/or onset of type 1 diabetes.

Conclusion

[0241] These results provide the first evidence of a causal link between influenza virus infection and the development of type 1 diabetes and/or pancreatitis. This causal link between infection and type 1 diabetes and/or pancreatitis provides various therapeutic, prophylactic and diagnostic opportunities.

[0242] The above description of preferred embodiments of the invention has been presented by way of illustration and example for purposes of clarity and understanding. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that many changes and modifications may be made thereto without departing from the spirit of the invention. It is intended that the scope of the invention be defined by the appended claims and their equivalents.

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