Immune responses after local administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs

Veterinary Immunology and Immunopathology 109 (2006) 209–217 www.elsevier.com/locate/vetimm

Immune responses after local administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs Anne-Marie Torche´ a,b,*, Mireille Le Dimna b, Pascal Le Corre a, Alain Mesple`de b, Se´bastien Le Gal b, Roland Cariolet c, Marie-Fre´de´rique Le Potier b a

Laboratoire de Pharmacie Gale´nique, Biopharmacie et Pharmacie Clinique, UPRES EA 3892, Faculte´ des Sciences Pharmaceutiques et Biologiques, Universite´ de Rennes I - 2, Avenue du Professeur Le´on Bernard, 35043 Rennes cedex, France b Unite´ Virologie, Immunologie Porcine, UPRES EA 3892, Agence Franc¸aise de Se´curite´ Sanitaire des Aliments, Les Croix – B.P. 53, 22440 Ploufragan, France c Service de Production de Porcs Assainis et d’Expe´rimentation, Agence Franc¸aise de Se´curite´ Sanitaire des Aliments, Les Croix – B.P. 53, 22440 Ploufragan, France Received 3 January 2005; received in revised form 25 July 2005; accepted 11 August 2005

Abstract Oral vaccination of large animals using PLGA MS (poly(D,L-lactide-co-glycolide)microspheres) appeared to be more challenging than immunization of mice. The purpose of this study was to deliver to GALT an immunogenic model protein (IgY), free or encapsulated by spray-drying in PLGA MS, and to evaluate systemic immune response in SPF Large White pigs. Pigs were surgically processed for local administration of IgY in three sets of experiments. In two sets of experiments, administration was locally performed in temporary ligatured intestinal segments, in jejunal Peyer’s patches and in mesenteric lymph nodes. In the third experiment, pigs received IgY via an intestinal cannula. Total IgY-specific antibodies were detected in the sera of pigs after a single local immunization, but not in the sera of cannulated pigs. The study of IgG1 and IgG2 isotypes indicated that PLGA MS are able to elicit a combined serum IgG2/G1 response with a predominance of IgG1 response when locally administered. PLGA MS can be a potential oral delivery system for antigen but our results underlined the difficulty to immunize large animals like pigs. Transposition of data between small and large animals appears to be complex and suggests that physiological features need to be considered to increase intestinal availability of oral encapsulated vaccines. # 2005 Elsevier B.V. All rights reserved. Keywords: Pig; Gut-associated lymphoid tissue; Local administration; PLGA microspheres; Immune response

Abbreviations: GALT, gut-associated lymphoid tissue; PP, Peyer’s patches; PLGA MS, poly(D,L-lactide-co-glycolide)microspheres; Specific Ig, total anti-IgY antibodies; IgY, avian immunoglobulin Y; IM, intramuscular; jPP, jejunal Peyer’s patches; jPP-E, administration in jPP after enterectomy; jPP-IW, administration in jPP through intestinal wall; MLN, mesenteric lymph nodes; SPF, specified pathogen-free * Corresponding author. Tel.: +33 2 23 23 49 50; fax: +33 2 23 23 48 46. E-mail address: [email protected] (A.-M. Torche´). 0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.08.016

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1. Introduction Mucosal tissues of the gastrointestinal, respiratory and urogenital tracts play a key role in the organism since they are both, the gateway and the first line of defence against invasion by microorganisms. Protection requires effective vaccination that stimulates immune responses at the mucosal tissue (Brandtzaeg et al., 1997). Thus, there is a great deal of interest in the development of mucosal vaccine strategies (Chen, 2000). For this purpose, poly(D,L-lactide-co-glycolide)microspheres (PLGA MS) designed for the encapsulation of soluble antigens (Ag) are intensively investigated. Most of the studies focus on oral administration of vaccines since: (i) the oral route appears to be easy to handle in humans and animals; (ii) the gut-associated lymphoid tissues (GALT) represent the largest mucosal area. GALT is characterized by lymphoid follicles, named Peyer’s patches (PP). Pig presents isolated PP in the jejunum (jPP) and a continuous PP in the terminal ileum (iPP) (Pabst et al., 1988). jPP have a preferential function as sites for antigen sampling, frequency of Ig secreting cells and induction of intestinal immune response (Barman et al., 1997; Pabst and Rothkotter, 1999; Bianchi et al., 1999). PP are specifically covered by specialized epithelial cells, M cells. These cells allow the transport of Ag across the follicle-associated epithelium (FAE) to the antigen-processing cells of the underlying dome region with subsequent stimulation of B and T lymphocytes (Gebert, 1997; Kraehenbuhl et al., 1997; Neutra, 1999). Transcytosis activity has been demonstrated for various polymeric nano- or microparticles (Jepson et al., 1993a; Ermark et al., 1995; Smith et al., 1995) in many species including pig (Torche´ et al., 2000). Different attempts were performed to demonstrate the potentiality of PLGA MS as vaccine adjuvant systems in order to induce both systemic and local immune responses following oral immunization. Experiments with antigens entrapped in PLGA microspheres have demonstrated that immunization boosts the systemic antibody response, enhances mucosal IgA responses in mice following oral or intragastric administration (Conway et al., 2001; Jung et al., 2001; Fattal et al., 2002; Yeh et al., 2002; Carcaboso et al., 2003) and is able of priming cellular (cytotoxic T lymphocyte) immune

responses in vivo (Maloy et al., 1994; Partidos et al., 1999). Most vaccination experiments were successfully performed on small animals like mice. Even if the number of published data is smaller, it appeared clearly more difficult to obtain the same results with large animals. To date, these delivery systems remain unsuccessful (Felder et al., 2000) or are largely untried for oral vaccine delivery in large animals (Mutwiri et al., 2002; Lin et al., 2003). The apparent discrepancy between results from small and large animals raises the problem of transposition and suggests that additional barrier may impede the local trafficking of MS throughout the GALT. In order to challenge this assumption, we have investigated the systemic immune response (total immunoglobulins, IgA, IgG1 and IgG2 isotypes) after administration of a model antigen (IgY) to pigs, as solution or encapsulated in PLGA MS, at different steps of Ag trafficking in the GALT. A surgical experimental model was set up to locally deliver IgYat different GALT locations, i.e. in intestinal lumen, in mesenteric lymph nodes or within Peyer’s patches.

2. Materials and methods 2.1. Preparation of PLGA microspheres containing IgY Avian immunoglobulin Y (IgY 160 kD) was isolated from chicken egg yolks by sodium precipitation. Two milliliters of aqueous solution containing IgY was emulsified by an ultrasonic probe with a 3% (w/v) solution of PLGA dissolved in dichloromethan (40 ml, RPE-ACS, Carlo Erba Reactifs) in a thermostated beaker (2 8C) to produce a stable W/O nanoemulsion. PLGA MS were subsequently formed by spray-drying the nanoemulsion at a rate of 2 ml/ min under the following conditions: inlet air temperature 50 8C; aspiration setting 100%; pump control 15%; air flow rate 500 NL/h (Mini-Bu¨chi 190). Two formulations of PLGA MS (A and B) were prepared by a spray-drying method. Formulation A was prepared using an organic phase containing Phusis1 75:25 poly(L-lactide-co-glycolide) and 0.05% (w/v) phosphatidylcholine. IgY was diluted in PBS at a concentration of 4.5 mg/ml (w/v).

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Formulation B was prepared using Phusis1 75:25 poly(D,L-lactide-co-glycolide). The aqueous phase contained IgY in PBS pH 7.4 at a concentration of 7.2 mg/ml (w/v) and 1% mannitol (w/v). 2.2. Characterization of PLGA microspheres The protein content of PLGA MS was determined by BCA assay (Pierce) after recovering IgY included in PLGA MS by acetonitrile precipitation. Encapsulation efficiency (%) was expressed by relating observed IgY entrapment to theoretical IgY quantity used. In vitro IgY release kinetics was performed and IgY was measured (micro-BCA) in collected supernatants after incubation of PLGA MS in PBS/azide (pH 7.4) at 37 8C. PLGA MS size was determined by laser diffractometry (Mastersizer S, Malvern Instruments) and expressed as mean volume diameter (D 4.3 mm). Structural integrity and immunogenicity of entrapped IgY was assessed by 4–6% SDS–PAGE electrophoresis and Western blotting. The blot was incubated with 1:1000 diluted alkaline phosphatase-conjugated goat anti-IgY IgG (Jackson Immunoresearch).

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In experiment 3, IgY was administered through a PVC/silicon cannula positioned by a surgical technique described by Cuche and Malbert (1999). Cannula was put into the duodenum and locally fixed with sutures. The end of cannula was exteriorised through an incision in the abdominal wall. The abdomen was sutured and the cannula was fixed on the back of the pig. Pigs recovered quickly, showing no clinical signs and no difference in weight gain. All pigs used in the study received an antibiotic cocktail of penicillin–streptomycin for 5 days to avoid infection. 2.4.2. Antigen preparation and administration Soluble IgY was diluted in sterile PBS prior to administration. PLGA MS, formulation A for experiments 1 and 3 and formulation B for experiment 2, were accurately weighted and suspended in sterile PBS immediately before immunization. IgY, free or encapsulated, was delivered as a single dose to pigs in experiments 1 and 2. In experiment 3, pigs were primed 3 weeks after the surgical procedure and then boosted 28 days after the first administration. Soluble BSA (7 mg) or blank MS were used as negative control antigen.

2.3. Animals Specified pathogen-free (SPF) Large White pigs (n = 22) were used in this study. The pigs were housed and reared in protected facilities and fed ad libitum. All animal experiments described below have been performed in the AFSSA Ploufragan facilities (registration No. B-22-745-1, authorization number: 22-25). 2.4. Immunization protocols 2.4.1. Surgical procedures In the three sets of experiments, except pigs that received intramuscular administration (IM), pigs were locally administered IgY (free or encapsulated) or negative control (BSA solution and blank microspheres) after a surgical procedure. Pigs were fasted 24 h before surgery. Each pig was intravenously anaesthetized with tiletamine/zolazepam/ketamine/xylazine. Then, the abdomen was aseptically opened. After local injection of the antigen as described below (experiments 1 and 2), muscle and skin of the abdomen were closed using a nylon suture.

2.4.3. Sample collection Blood samples were weekly collected during the time period of the assay (experiment 1: 56 days, experiment 2: 61 days). Samples were centrifuged and serum was collected and stored at 20 8C. 2.4.4. Experiments IgY was delivered into different compartments of the GALT. The three sets of experiments performed in this study are summarized in Table 1. Administration in jejunal Peyer’s patches (jPP) were performed either directly through the intestinal wall (jPP-IW) since PP were visible on the serosal surface or after enterectomy (jPP-E). Administration in mesenteric lymph nodes (MLN) was performed directly in draining MLN. Administration in intestinal lumen was performed directly via the cannula or in an intestinal segment containing one or two isolated PP ligatured and filled with 10 mg of IgY, either free or encapsulated. The ligature was maintained for 15 min in order to increase the time contact between IgY with GALT.

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Table 1 Synoptic of immunization experiments performed on SPF pigs Route

Number of pigs

Intramuscular

Age (weeks)

IgY (mg/dose)

Volume (ml)

4 3

1

Solution Formulation A Formulation B

Exp 1

1.5 5 1.5 5 1.5 5 1.5 5 1.5 5

Solution Formulation A Solution Formulation A BSA solution Blank MS Solution Formulation A Solution Formulation B

Exp 1

Solution Formulation B Formulation B Solution Formulation A

2

7 In situ Mesenteric lymph node Jejunal Peyer’s patches—intestinal wall

2

4

7

4

4

7 0

Jejunal Peyer’s patches—enterectomy

2

4

7

Jejunal Peyer’s patches—intestinal wall

2

7

10

3

7

10

9

2 4

4

10

20

Intestinal lumen Jejunal segment Ileal segment Cannula

2.5. Total Anti-IgY antibody detection Anti-IgY antibodies (specific Ig) were measured by conventional indirect ELISA. Microtitre plates (NuncImmuno Plate1 FP96 Maxisorp) were coated with 100 ml per well of 0.5 mg/ml IgY in carbonate buffer (pH 9.6) and incubated 1 h at 37 8C, then placed overnight at +4 8C. Plates were washed with PBS containing 0.1% Tween-20 (PBST) and blocked for 1 h at 37 8C with 200 ml PBST containing 2% (w/v) non-fat dry milk (Bio-Rad). Serum samples and the negative control (SPF pig serum) were diluted 1:100 in PBS, and 100 ml of each sample were added to each well. The positive control (hyperimmune pig serum) was serially diluted from 1:1000 to 1,000,000. Plates were incubated for 1 h at 37 8C and washed three times with PBST. Hundred microliters of HRPconjugated polyclonal rabbit anti swine immunoglobulins (P0164; Dako) diluted 1:1000 in blocking buffer were added to each well. Plates were incubated for 30 min at 37 8C and washed three times with PBST. Then 100 ml of chromogen (orthophenylenediamine and hydrogen peroxide in citrate buffer (pH 5.0) were added to each well and incubated at 37 8C for color development. The reaction was stopped after 6 min by

Antigen

Exp 2

Exp 1

Exp 1 Exp 2

Exp 2

Exp 3

addition of 50 ml of 1N H2SO4. Absorbance values (A) for each sample were read at 490 nm with a plate reader (Dynatech). ELISA was done in triplicate. Results are expressed as positive percentage of hyperimmune serum, diluted 10,000, calculated as follows: Aexperimental serum Anegative control  100 Ahyperimmune serum Anegative control A positive threshold (25%) was established for specific total Ig. 2.6. Anti-IgY isotypes detection Antibody isotype analysis of pig sera was performed using the same ELISA with some modifications. Microtitre plates were coated with 100 ml per well of a 2.5 mg/ml IgY in carbonate– bicarbonate buffer (pH 9.6). After identical steps of washings, blocking and serum samples (dilution 1:100) incubation, monoclonal antibodies (Id-DLO, Lelystadt), anti-swine IgA (mabaSw 27.9.1), antiswine IgG1 (mabaSw 23.4.91) and anti-swine IgG2 (mabaSw 34.1.1a) were added to the wells at the dilution of 1:500 in PBST. The plates were incubated for 1 h at 37 8C and washed three times with PBST.

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100 ml of HRP-conjugated polyclonal rabbit anti mouse immunoglobulins (P0260; Dako) diluted 1:500 in blocking buffer was added to each well. The plates were incubated for 30 min at 37 8C and washed three times with PBST. After addition of the substrate as previously described, the reaction was stopped after 3 min of incubation by the addition of 50 ml of 1N H2SO4. ELISA was done in triplicate. Results are expressed as described above. A positive threshold (10%) was established for isotypes.

3. Results and discussion 3.1. Characterization of PLGA MS The average diameter for formulation A and formulation B was 4.2 mm and 5.6 mm, respectively, with a narrow size distribution and a lack of aggregates in the suspension. The encapsulation efficiency of IgY for both formulations was near 90% (w/w). After a small initial burst release after 1 h incubation (6.1% for formulation A and 9.3% for formulation B), a zero-order release was observed with an increase rate above 5–7 days from 0.4 to 2.4%/day for formulation A and from 1.0 to 4.9%/day for formulation B. The release profiles obtained between the two formulations were different since formulation B displayed substantially higher release rate (Fig. 1). This may be explained by the differences between the two formulations (i.e. phosphatidylcholine and mannitol). It could be suggested that IgY was differently localized in formulations A and B and/or each formulation could present distinct water uptake rates influencing the release of IgY. No additional bands indicating the presence of IgY aggregates or fragments were observed on SDS– PAGE gel and Western blot confirmed that the immunogenicity of IgY was not altered during PLGA MS fabrication (data not shown). 3.2. Induction of systemic IgY-specific antibody responses 3.2.1. Induction of systemic IgY-specific antibody responses after intramuscular administration Following a single IM administration, free IgY induced higher serum specific Ig response compared

Fig. 1. Cumulative in vitro release profiles of IgY from PLGA MS formulations A (*) and B (&). Values are represented as mean (standard deviation).

to encapsulated IgY (Fig. 2a). Moreover, formulation B could not induce specific immune responses so effectively than formulation A. This observation suggests that the properties of PLGA MS influence the induction of systemic immune response. A rapid clearance of PLGA MS and/or difference in IgY release, as we have seen between formulations A and B, may explain this observation. 3.2.2. Induction of systemic IgY-specific antibody responses after local administration Oral vaccination with PLGA MS appears difficult to be extrapolated from small laboratory animals to large animals. Since we have met difficulties to induce immune response with pigs assayed under a conventional experimental procedure (i.e. oral gavage), we have decided to focus on local delivery of PLGA MS in the compartments where such particles could be found after oral administration. The Fig. 2b shows the immune response after administration of IgY in jejunal Peyer’s patches

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Fig. 2. Induction of total anti-IgY immunoglobulins in pigs immunized with free IgY (open symbol) or with PLGA MS (full symbol) containing IgY. Experiment 1/formulation A (plain line). Experiment 2/formulation B (dashed line). One curve corresponds to one pig. (a) Intramuscular administration (^ ^); (b) direct administration in jejunal PP through the intestinal wall (& &); (c) direct administration in jejunal PP through the FAE after enterectomy (~ ~) and direct administration in MLN (* *); and (d) instillation in ligatured segment in intestinal lumen (~ ~ jejunum; * ileum).

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through the intestinal wall (jPP-IW) (experiments 1 and 2). Ig production was similar until about day 40. Then, in experiment 1, Ig production still increased by opposition to experiment 2. This discrepancy between experiments 1 and 2 cannot only be attributed to the use of two PLGA MS formulations since free IgY has led to different Ig levels. The influence of pig age (4 and 7 weeks in experiments 1 and 2, respectively) may explain such discrepancy and should be further studied. Negative controls administered in jPP-IW led to positive percentage lower than the threshold (BSA  18.4%, blank MS  11.4%). Local administration was also performed by enterectomy (jPP-E), a more invasive method. Specific Ig response obtained with PLGA MS (formulation A) was three-fold higher than that obtained with free IgY (Fig. 2c) and was different, almost two-fold, to the immune response induced after administration through the intestinal wall (Fig. 2b). It could be suggested that the depth within PP where IgY was delivered was not the same by the two methods since PLGA MS formulation used and pig age were the same. PLGA MS, in our size range, can be translocated to mesenteric lymph nodes (MLN) and subsequently to the spleen (Carr et al., 1996). MLN contain a large population of lymphocytes and antigen-presenting cells, allowing a good immune response to be expected after local induction. Direct administration of PLGA MS in MLN induced a stronger systemic immune response, almost two-fold higher than free IgY (Fig. 2c). Specific Ig production was sustained or increased during all the period studied. The present result illustrated the potential of PLGA MS as adjuvant. Serum immune responses following instillation into ligatured segments of IgY, free or encapsulated, are presented in Fig. 2d. PLGA MS appeared to elicit relatively close specific Ig levels when instillated in jejunal and ileal segments. Free IgY instilled in intestinal lumen was able to induce serum Ig, suggesting that the time contact with enzymatic content of the intestine was too short for a complete denaturation. Since instillation of PLGA MS in intestinal ligatured segments has elicited a systemic immune response, we have investigated the local delivery of

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PLGA MS in intestinal lumen in cannulated pigs without any intestinal ligatures. Previously, formulation A appeared to elicit better immune responses than formulation B, and hence was employed in experiment 3. However, no specific Ig were detected, despite of the boost administration at day 28. The comparison of results from IgY administration in a temporary ligatured intestinal segment and via a cannula gave us interesting informations. PLGA MS not only gives rise to significant antibody titres when instilled in a ligatured segment but also indicate the importance of residence time of PLGA MS for efficient uptake in lymphoid follicles and induction of immune response, since no Ig were dosed in cannulated pigs. We could suggest that PLGA MS were flushed by peristaltism and/or cleared by normal mucus clearance in faeces. Results strongly suggested that formulations, which can further increase the residence time in the intestine and subsequently the extent of particle uptake, are desirable. 3.2.3. Induction of systemic IgY-specific antibody subclasses IgA response was different according to the site of Ag administration and the vehicle, free or encapsulated (Fig. 3a). Intramuscular administration of PLGA MS induced similar IgA level than with free IgY. Production of serum IgA was greatly enhanced when PLGA MS (formulation A) were used in MLN and in PP after enterectomy compared to solution and was much higher than after IM administration. After instillation in ligatured intestinal segments, the slight IgA production was higher when PLGA MS were used. IgG1 appeared to be the main subclass in serum responses whatever the administration sites (Fig. 3b); moreover, specific IgG1 level remained stable after Ag administration (mean of positive percentages from day 20 to day 56 (experiment 1) or to day 61 (experiment 2): CV < 15%). After IM administration, IgG1 production was similar with formulations A and B. However, administration of PLGA MS seemed to have no influence on IgG1 production in all cases studied. After administration of PLGA MS in MLN and jPP-E, it should be noticed that a high level of IgG2 was observed compared to free IgY and by opposition to IM route (Fig. 3b). Instillation in ligatured segments led to small IgG2 levels, which were higher when PLGA MS were used.

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presentation to immunocompetent cells since isotype production was increase when PLGA MS were locally administered, i.e. intestinal lumen, PP and MLN. Accordingly, the effectiveness of PLGA MS to deliver antigen to PP depends on their ability to cross mucosal barriers and on appropriate release kinetics of the encapsulated antigen. Acknowledgements This work was financially supported by Direction Generale de l’Alimentation (DGAl), Ministe`re de l’Agriculture, de l’Alimentation, de la Peˆche et des Affaires rurales. We thank Dr C. Malbert (INRA Saint Gilles, France) for providing cannula. References

Fig. 3. (a) Anti-IgY serum IgA in pigs immunized with IgY in PLGA MS (filled bar) or in aqueous solution (open bar). Experiment 1 at days 0, 20, 36, 49 and 56 (black filled bar), experiment 2 at days 0, 20, 39, 53 and 61 (grey filled bar). (b) Anti-IgY serum IgG subclasses: IgG1 (open bar) and IgG2 (filled bar) in pigs immunized with IgY in PLGA MS or in aqueous solution. IgG1was expressed as a mean (positive percentage from day 20 to day 56 (experiment 1) or to day 61 (experiment 2)). Experiment 1 at days 0, 20, 36, 49 and 56 (black filled bar), experiment 2 at days 0, 20, 39, 53 and 61 (grey filled bar).

These combined serum IgG1/IgG2 responses could reflect Th1/Th2-like balance of immune responses. However, such functions of porcine isotypes have not been yet described in pig, only hypothesized (Piriou et al., 2003; Crawley and Wilkie, 2003).

4. Conclusion Despite of promising results obtained in mice, it appears much more difficult to obtain similar results with large animals like pigs. In this study, PLGA MS were able to induce a systemic immune response after intestinal administration (lumen–PP–MLN). However, the intensity of systemic total antibody level was different according to the site of administration highlighting the influence of diffusion barriers (i.e. luminal < PP < MLN). The study of isotypes, IgA, IgG1 and IgG2, indicated the influence of antigen

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