Research in Veterinary Science 95 (2013) 786–793
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Immunogenetic responses in calves to intranasal delivery of bovine respiratory syncytial virus (BRSV) epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles Owen V. Kavanagh a,c, Brian M. Adair a,b, Michael Welsh a,b, Bernadette Earley c,⇑ a b c
Department of Veterinary Science, Queen’s University Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland, UK Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland
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Article history: Received 19 April 2013 Accepted 23 June 2013
Keywords: Bovine respiratory syncytial virus BRSV Poly(lactide-co-glycolide) PLG G-attachment glycoprotein F-fusion glycoprotein Subunit vaccine Intranasal inoculation
a b s t r a c t Bovine respiratory syncytial virus (BRSV) is the principal aetiological agent of the bovine respiratory disease complex. A BRSV subunit vaccine candidate consisting of two synthetic peptides representing putative protective epitopes on BRSV surface glycoproteins in soluble form or encapsulated in poly(lactide-coglycolide) (PLG) microparticles were prepared. Calves (10 weeks old) with diminishing levels of BRSVspecific maternal antibody were intranasally administered a single dose of the different peptide formulations. Peptide-specific local immune responses (nasal secretion IgA), but not systemic humoral (serum IgG) or cellular responses (serum IFN-c), were generated by all forms of peptide. There was a significant reduction in occurrence of respiratory disease in the animals inoculated with all peptide formulations compared to animals given PBS alone. Furthermore no adverse effects were observed in any of the animals post vaccination. These results suggest that intranasal immunisation with the peptide subunit vaccine does induce an as yet unidentified protective immune response. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Bovine respiratory disease, and in particular calf enzootic pneumonia, is a major problem among cattle populations worldwide, in which intensive or semi-intensive calf rearing systems are employed. This disease, which can terminate fatally, affects both the dairy and beef industry and is the most important disease affecting calves in terms of animal welfare and agri-economics (Andrews, 1998; Fulton, 2009). In an Irish survey in a slatted unit containing 6399 beef cattle over a 6 month period respiratory disease was the most frequently recorded case of morbidity and mortality (Healy et al., 1993), and this observation is in agreement with reports from large feedlots in America (Jensen et al., 1976). In most cases it would appear that the primary infective agent is viral, producing respiratory tract damage that is subsequently extended by Mycoplasmas and secondary bacterial infections. Bovine respiratory syncytial virus (BRSV) infection is the major cause of respiratory disease in beef and dairy calves during the first year of life (Bryson et al., 1978; Barrett 2000).
⇑ Corresponding author. Tel.: +353 469061100; fax: +353 469026154. E-mail addresses:
[email protected] (O.V. Kavanagh), brian.adair@afbini. gov.uk (B.M. Adair),
[email protected] (M. Welsh), bernadette.earley@ teagasc.ie (B. Earley). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.06.023
Licenced conventional BRSV vaccines are administered parenterally but do not confer complete protection. Several reports indicate that modified-live vaccines may actually exacerbate the disease condition after natural infection (Gershwin et al., 1998; Schreiber et al., 2000; Kimman et al., 1989a). Inoculation with live BRSV via the intranasal route, unlike the intramuscular route confers protection against viral excretion in the presence or absence of immunosuppressive maternal antibodies (Kimman et al., 1989b). The protective effect of antibodies in the nasal secretions of calves against BRSV re-infection has been demonstrated (Mohanty et al., 1976). Another approach to improve the protective effect of BRSV vaccines is focused on development of subunit peptide vaccines that generate a protective immune response in the animal but lack the viral components that exacerbate disease conditions. The two major BRSV surface glycoproteins, the G attachment and F fusion protein, represent the major targets of the protective immune response. A synthetic peptide corresponding to a highly conserved region 174–187 of BRSV G glycoprotein with a Cys186 ? Ser substitution (G174–187) conferred complete protection in mice challenged with BRSV (Trudel et al., 1991). Furthermore, intramuscular immunisation of calves with this peptide coupled to a carrier protein efficiently induced antibodies capable of recognising both the parent G protein and the peptide. Following challenge with live
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BRSV, the immunised animals were efficiently protected against BRSV-associated pneumonia however no protection of the upper respiratory tract was observed (Bastien et al., 1997). The synthetic peptide sequence of the BRSV F protein chosen as the candidate subunit vaccine spanned the amino acid region 111–148 (F111–148). This choice is based on work carried out by Rankin et al. to identify T cell epitopes on the bovine F protein using overlapping synthetic peptides representing the entire F amino acid sequence (Rankin et al., 1995). In their study they found that the F protein contains four main T cell antigenic sites one of which, spanning residues 100–148, also includes the region where the precursor fusion protein (F0) is cleaved into its subunit components F1 and F2 as well as the proposed hydrophobic, fusigenic part of the fusion protein. This may be an immunoprotective epitope as Taylor et al. found that protection against the closely related HRSV infection correlated with fusion inhibition (Taylor et al., 1992). Therefore it may be possible to generate protection against BRSV by inducing an immune response against these two peptides. Peptide antigens administered at mucosal sites are poorly immunogenic however this problem can be overcome by administration within biodegradable polymeric microparticulate systems. These are believed to work by (1) increasing antigen uptake into the microfold M cells of mucosal-associated lymphoid tissue (Almeida and Alpar, 1996, Eyles et al., 1998) and consequently enhanced presentation in antigen-presenting cells (Tabata and Ikada, 1990), and, (2) the polymer poly(lactide-co-glycolide) appears to activate dendritic cell maturation possibly by providing a necessary danger signal, however the exact mechanism is not fully elucidated (Bennewitz and Babensee, 2005; Yoshida and Babensee, 2004). In this study we compared the immunogenicity of soluble and microencapsulated peptides (with or without covalent linkage to the carrier protein ovalbumin (OVA)) following i.n. administration in calves with declining BRSV-specific antibodies. The clinical and immunological status of the animals was also monitored throughout the study. 2. Materials and methods 2.1. Peptide synthesis and purification The synthetic peptides were prepared using FASTMOC chemistry. The sequence representing the amino acid region of the BRSV fusion protein from the RB94 (Rispoval vaccine) strain was 111-IPELIHYTRNSTKRFYGLMGKKRKRRFLGFLLGIGSAI148 (F111–148) (Rankin et al., 1995). The amino acid sequence 174-STCEGNLACLSLSK-187 with a Cys-Ser substitution at position 184 (G174–187) (Bastien et al., 1997) represents the corresponding region of the attachment protein of the BRSV strains 375 and Snook. Isolation of the synthetic product was achieved by diethyl ether precipitation. The peptides were purified using high-performance liquid chromatography (HPLC) lyophilised from 5% acetic acid and stored at 20 °C. 2.2. Conjugation of peptide to carrier molecule and microencapsulation Five mg of peptide was dissolved in 500 ll of distilled water and the pH was adjusted to between 4.5 and 5.0 with 0.1 M NaOH. Twenty milligram of water-soluble N-ethyl-N0 -(3-dimethylaminopropyl) Carbodiimide (EDC) (Sigma–Aldrich Co. Ltd., Poole, UK) was dissolved into 200 ll distilled water and then added to the peptide solution and stirred for 1 h at 4 °C. Three mg of carrier protein (OVA) was dissolved in 300 ll of distilled water and then added to the peptide/EDC solution and incubated at 37 °C for 4 h
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with gentle agitation. The reaction mixture was dialysed in distilled water to remove the EDC and the protein complex was lyophilised and stored at 20 °C until use. Free or conjugated peptide was encapsulated in microparticles consisting of poly(lactide-co-glycolide) polymer with a molecular weight of 40–75 kDa and a lactide/glycolide ratio of 50/50 (Sigma–Aldrich Co. Ltd., Poole, UK) using a water-in-oil-in-water (w/ o/w) protocol as described previously (Kavanagh et al., 2003). 2.3. Microparticle characterisation The surface morphology and mean size of the microparticles was determined by Scanning Electron Microscopy (SEM) and flow cytometric analysis, respectively. Samples were prepared by applying a thin film of lyophilised powder from each microparticulate formulation onto aluminium stubs, the upper surfaces of which were applied layers of conductive double sided tape, (Agar Scientific Ltd., Essex, UK), to aid adhesion. Specimens were stored in a dessicator prior to sputter coating with approximately 20 nm of gold using a Polaron E5100 series II ‘cool’ sputter coater (Polaron Equipment Ltd., Hertfordshire, UK). The specimen stubs were then stored in a dessicator prior to SEM examination using a JEOL 35CF Scanning Electron Microscope (JEOL (UK) Ltd., Welwyn Garden City, Herts., England). Flow cytometric analysis of the microparticles was performed using a FACS Vantage (Becton Dickinson, La Jolla, CA) equipped with an Innova Enterprise ion laser (Coherent Laser Group, San Jose, CA). The size range distribution of microparticles suspended in PBS (0.1 mg/ ml) was estimated on the basis of forward and orthogonal light scatter in comparison to 2.49 lm (Red Nile beads: Becton Dickinson) and 10 lm (Fluorospheres: Coulter Corporation, Miami, Florida) diameter flow cytometric alignment beads. The content of encapsulated peptide was determined using a bicinchoninic acid (BCA) protein determination assay (Pierce & Warriner Ltd., Cheshire, UK) after dissolution of approximately 10 mg of microparticles in 600 ll 0.1 M NaOH (BDH Laboratory Supplies, Poole, UK) containing 5% sodium dodecyl sulphate (BDH). The extraction was stopped after 4 h at 37 °C in an orbital shaker (Gallenkamp, Loughborough, UK.) with 500 ll 0.1 M HCl, the solution was centrifuged and the supernatant removed and stored at 20 °C until analysis. 2.4. Animals This study was conducted at the Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Meath, Ireland. All animal procedures performed in this study were conducted under experimental licence from the Irish Department of Health and Children in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendment of Cruelty to Animals Act 1876) Regulation 2002 and 2005. Sixty-five mart purchased Holstein/Friesian calves were approximately 18 days of age on arrival at the research centre. The calves were individually penned (pen size of 1.55 m2, floor area of 2.16 m2 and cubic air capacity of 7.4 m3/calf) in a naturally ventilated calf shed. The calves received an individual allowance of 25 kg of milk replacer powder offered warm at 38 °C by bucket during the first 42 days and had ad libitum access to a concentrate ration throughout the trial period. From day 43 onwards, the calves had ad libitum access to a concentrate ration and roughage (straw) throughout the experimental period. Clean fresh water was available at all times. At 10 weeks of age, the calves were allocated randomly and received one of the following 5 treatments (13 per treatment); (1) soluble F-peptide (sF) and soluble G-peptide (sG) in phosphate buffer saline; (2) soluble peptide microencapsulated in poly(lactideco-glycolide) (PLG-F) and (PLG-G); (3) peptide coupled to
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ovalbumin microencapsulated in PLG (PLG-OVA–G) and (PLG-OVAF); (4) (PLG-OVA–G) and (PLG-OVA-F) admixed with 20 lg cholera toxin for positive control and (5) phosphate buffered saline negative control. Immediately before nasal administration the freezedried microspheres containing the encapsulated peptides were suspended in 2 ml of sterile physiological PBS and administered to individual calves in a syringe fitted with an intranasal applicator.
2.5. Collection of biological fluids Nasal mucosa washings and blood samples were collected the morning before inoculation (pre-immune) and on day 28 and day 77 post- inoculation. Nasal mucus was analysed for peptide-specific secretory IgA antibody levels in response to inoculation. Blood samples were collected by jugular venipuncture and left overnight at 37 °C to separate the serum from plasma. The serum was obtained by centrifugation at 800g for 5 min and stored at 20 °C. Nasal mucosa washings were collected by inserting a sponge into the animals’ nostril for 10 min. Nasal secretions were centrifuged at 800g for 5 min at 4 °C to remove aggregated mucus and stored at 20 °C.
2.8. Preparation of positive control samples for standardisation of assays Independent of the immunogenicity study, two BRSV-negative calves were intramuscularly immunised with 0.5 mg of each peptide coupled to ovalbumin mixed in MPL adjuvant (Sigma) on 3 occasions 3 weeks apart. Three weeks after the third inoculation blood samples were taken by venipuncture and the sera tested for peptide-specific IgG. This hyperimmune serum was used as a positive reference sample for the peptide-specific IgG ELISA at a dilution of 1/100. Parenteral immunisation of these two calves with peptide and antigen failed to generate any peptide specific IgA in nasal secretions so a series of nasal secretions taken from animals used in this study, before and after inoculation with various formulations of microencapsulated peptide, were analysed for peptide-specific IgA using the ELISA (see below). Pooled samples of approximately 3 ml of nasal washes, which contained high levels (OD > 1.0) of either peptide–specific IgA, were made. These positive pooled samples were diluted in 12 ml of PBS-T containing 3% BSA (Sigma) and 0.02% sodium azide (BDH Laboratory Supplies, Poole, UK). Aliquots (3 ml) were prepared and stored at 20 °C until required. 2.9. Detection of peptide specific antibody
2.6. Immunoglobulin determination Serum immunoglobulins (IgG1) were measured quantitatively by single radial immunodiffusion (sRID) (Mancini et al., 1965) and calculated via an internal immunoglobulin (Ig) standard (BINDARID, NANORID kits. The Binding site Ltd., R&D, Birmingham, UK). The zinc sulphate turbidity (ZST) test was performed on all serum samples at 20 °C with turbidity readings carried out at 520 nm using a spectrophotometer (McEwan et al., 1970).
2.7. Measurement of BRSV-specific maternal antibodies A commercially available indirect ELISA kit (Svanovir™, Svanova Biotech, Uppsala, Sweden) was used to measure the levels of maternally-derived BRSV-specific antibody in sera of the calves used in this study. The solid phase consisted of non-infectious BRSV antigen on a Polysorb (Nunclon, Nunc, Denmark) microtiter wells. To account for non-specific binding of sera constituents to non-viral components, wells without BRSV antigen were used as a negative control. The procedure is described in detail elsewhere (Westenbrink et al., 1989). PBS-T buffer (100 ll) was added to each well used for sera and reference samples. 4 ll of each serum sample was added to appropriate wells coated with BRSV and negative control antigen. BRSV positive and negative reference sera (4 ll) were added to the positive and negative wells and plates were incubated for 1 h at 37 °C. After thorough washing with PBS-T 100 ll of antibovine IgG horse-radish peroxidase was added to each well and incubate at 37 °C for 1 h. Plates were washed thoroughly and 100 ll of substrate solution was added to each well and incubate for 10 min at room temperature. The reaction was stopped by adding 50 ll of the stop solution (0.1 M H2SO4) and the OD values of samples and reference wells were measure at 450 nm using an ELISA plate reader (Spectra II, SLT Labinstruments, Austria). The optical density values in wells coated with BRSV were corrected by subtracting the OD values of the corresponding wells containing control antigen. Serum samples were considered positive if the corrected OD value at 450 nm > 0.2. The corrected OD values were expressed as a ratio of the positive reference OD values from the corresponding plate.
The presence of BRSV peptide-specific IgA and IgG antibody in nasal washings and sera, respectively, was detected using an ELISA. Unless stated otherwise, the final volume in each well was 100 ll. 10 lg/ml of F111–148 or 1.0 lg/ml G174–187 were used as fixed antigen on Immulon type 2HB (Dynex Technologies Inc., Chantilly, VA), or, Maxisorp (Nunclon, Nunc, Denmark) 96 well plates, respectively. The peptide was dried onto the plates overnight at 37 °C. The plates were washed five times with PBS containing 0.05% Tween 20 (PBS-T) using a wash bottle. Nasal mucosa samples were diluted 1/5 in 5% non-fat dried milk (NFDM) and sera diluted 1/100 in PBS-T + 5% NFDM and were incubated for 1 h at 37 °C. The plates were washed 5 times as before and mouse anti-bovine IgA (Serotec Ltd., Oxford, UK) for the nasal secretions, or rabbit anti-bovine IgG horse radish peroxidase conjugate (Sigma), for sera IgG, at a dilution of 1/300 and 1/10,000, respectively, in PBS-T containing 5% NFDM was added to the wells for 1 h at 37 °C. Plates were again washed as before and goat antimouse IgG horseradish peroxidase conjugate diluted 1/5000 (Zymed, Cambridge Bioscience, Cambridge, UK) in PBS-T was added to the each well of the nasal IgA plates only, and incubated overnight at 4 °C. The plates were washed thoroughly with PBS-T and 100 ll of TMB substrate (Chemicon International Ltd., Harrow, UK) was added to each well and the colorigenic reaction was stopped after 5 min at ambient temperature with 50 ll 0.1 M HCl. Optical density (OD) values were read at 450 nm using an ELISA reader (Spectra II, SLT Labinstruments). The OD values were standardised using nasal wash positive pool containing peptidespecific IgA or peptide hyperimmune bovine sera. The OD values for the IgA ELISA were expressed as a ratio of a positive control serum on each plate. Furthermore, to allow for differences in nasal wash protein content, these results were normalised to total protein concentration for each sample, as determined by the BCA assay (Pierce & Warriner Ltd., Cheshire, UK). 2.10. Production of interferon-c Twenty ml of blood was collected into evacuated blood collecting tubes containing heparin (Vacutainer Systems, Becton Dickinson Systems Europe, Meylan Cedex, France) at day 28 post-inoculation. The blood samples were evenly mixed before aliquoting on a roller rocker for 10 min at room temperature.
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1.5 ml of whole blood was added in triplicate to 24 well trays (Iwaki Microplate, Scitech Division, Asahi Techo Glass, Japan) and incubated with either 25 lg/ml of F111–148 or 12.5 lg/ml of G174–187. Whole bloods were incubated with 15 lg/ml of concanavalin A (Con A) or sterile PBS as a positive and negative control of interferon-c production, respectively. All blood samples were incubated for 72 h at 37 °C/5% CO2. The plasma was harvested by centrifuging the 24-well plates at 500 x g for 10 min at room temperature. The plasma supernatant from each well was removed and stored at 20 °C before assaying. A commercially available sandwich ELISA kit (Bovigam™, CSL Veterinary Ltd., Victoria, Australia) was used for the quantification of bovine interferon-c in blood samples. All supernatant samples and reagents were equilibrated to room temperature before use. 50 ll of each supernatant sample (test & controls) was incubated in duplicate with 50 ll of Green diluent in the ELISA plates provided for 1 h at 37 °C/5% CO2. The plates were then emptied and washed six times with the wash buffer provided. 100 ll of anti-bovine conjugate freshly reconstituted in blue diluent was incubated for 1 h at 37 °C/5% CO2. The plates were washed as before and 100 ll of enzyme substrate solution was added to the wells and incubated for 10 min at room temperature. The colorigenic reaction was stopped by adding 50 ll of stop solution to each well and the optical density of the substrate was read at 450 nm using an ELISA plate reader (SLT Labinstruments). 2.11. Clinical status Calves were weighed before assignment to treatment, and on day 7, 14, 21, 28, 35, 42, 48, 56, 63, post-immunisation to determine the average daily gain (ADG). Calves with a rectal temperature of P40 °C and clinical signs of respiratory disease were administered antibiotic for the treatment of clinical symptoms (defined individually for each animal). 3. Statistical analysis
Fig. 1. Characterisation of lyophilised poly(DL-lactide-co-glycolide) microparticles containing BRSV peptide antigen. A 50/50 combination of microparticles containing the BRSV peptides G174–187 and F111–148, coupled to ovalbumin, prepared in sterile PBS were used as an example of all microparticles preparations. Characterisation was done using (A) scanning electron micrograph of microparticles (size bar in the bottom right corner of the micrograph represents 1 lm) and (B) flow cytometric analysis of the size range distribution of the PLG microparticles. The microparticles were analysed at a concentration of 0.1 mg/ml and the size range was determined using alignment beads of known size (R1 = 2.49 lm; R2 = 10 lm).
The data was analysed with GraphPad PrismÒ (version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com) software using analysis of variance procedure and if a significant difference was observed, Duncan’s multiple range test was applied to determine statistical differences between treatments. P < 0.05 was considered statistically significant. 4. Results 4.1. Microparticle characterisation The surface morphology of an equal mix of PLG-OVA-F and PLGOVA-G freeze-dried particles were used as a representative for all particle formulations. The particles were visualised using a Scanning Electron Microscope (Figure 1A). The particles were spherical in shape with a relatively smooth surface. Flow cytometric analysis was used to estimate the size range distribution of the particles in comparison to flow cytometric alignment beads of known size. Fig. 1B shows the size distribution of the particles, indicating that virtually all of the particles were
