Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in...

Journal of Controlled Release 85 (2002) 191–202www.elsevier.com/ locate/ jconrel

M ucosal immune responses following oral immunization withrotavirus antigens encapsulated in alginate microspheres

a,b c a c ,1 a cB. Kim , T. Bowersock , P. Griebel , A. Kidane , L.A. Babiuk , M. Sanchez ,a a a ,*S. Attah-Poku , R.S. Kaushik , G.K. Mutwiri

aVeterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan,Canada S7N 5E3

bNational Veterinary Research Institute, 480 Anyong, Kyunggi-Do, South KoreacPharmacia Animal Health, 7000 Portage Road, Kalamazoo, MI 49001-0199,USA

Received 12 December 2001; accepted 10 April 2002

Abstract

Availability of effective oral vaccine delivery vehicles should contribute to the success of oral immunization in domesticanimals. To achieve this goal, we evaluated alginate microspheres for their capacity to induce mucosal immune responsesfollowing oral and enteric immunizations. Mice were immunized with either live porcine rotavirus (PRV) or its recombinantVP6 protein, encapsulated in alginate microspheres or unencapsulated. VP6-specific IgG (but no IgA) antibodies weredetected in the sera of mice after a single intraperitoneal (i.p.) immunization with either VP6 in Incomplete Freund’s adjuvant(VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freund’s adjuvant (PRV-IFA). Incontrast, VP6-specific IgA (but no IgG) was detected in culture supernatants of mesenteric lymph nodes from miceimmunized i.p. with either VP6-IFA or with PRV-IFA. Oral immunization with VP6-MS induced the highest level ofVP6-specific fecal IgA antibody, similar to responses induced by oral immunization with live PRV. Furthermore, theVP6-specific fecal IgA could be boosted by a secondary i.p. immunization with VP6. Further experiments were performed ina sheep intestinal ‘loop’ model to evaluate uptake of microspheres by Peyer’s patches. Microspheres containing colloidalcarbon were specifically bound and transported by follicle-associated epithelium of Peyer’s patches. Additionally, mucosalimmune responses were detected following enteric immunization with porcine serum albumin (PSA) encapsulated in alginatemicrospheres. Our results confirm that alginate microspheres are an effective oral delivery vehicle for protein antigens andintestinal IgA antibody responses are induced by antigens encapsulated in alginate microspheres without any additionalmucosal adjuvant. These investigations confirm that alginate microspheres have the potential as an effective delivery vehiclefor oral immunization of ruminants. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Mucosal immunity; Oral vaccine delivery; Alginate microspheres

1 . Introduction*Corresponding author. Tel.:11-306-96-67472; fax;11-306-

96-67478.Vaccination is the most cost-effective approach toE-mail address: [email protected](G.K. Mutwiri).

1Current address: Shire Laboratories, Rockville, MD, USA. prevent economic losses and morbidity caused by

0168-3659/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 02 )00280-8

mailto:[email protected]

192 B. Kim et al. / Journal of Controlled Release 85 (2002) 191–202

infectious diseases in animals. Most of the vaccines tion can involve the use of organic solvents. Theseavailable today are injected parenterally and are not solvents, along with the internal pH of the degradingeffective in inducing immunity at mucosal surfaces. PLG microparticles may alter antigenic epitopes andUnfortunately, it is through these mucosal surfaces hence compromise immunogenicity [11].that the majority of pathogens invade the body. There are relatively few reports of using micro-There is compelling evidence indicating that mucosal spheres made from sodium alginate (kelp), a natu-immunity is effectively induced when vaccines are rally occurring gelling polysaccharide that is exten-delivered to the mucosa-associated lymphoid tissue sively used in the food industry as a stabilizer and(MALT) located at mucosal surfaces [1–3]. Thus, thickening agent. Sodium alginate is water soluble,there is a need for effective mucosal vaccine delivery and polymerizes into a solid matrix upon contactif better disease protection against mucosal patho- with divalent cations [12]. Procedures for formulat-gens is to be achieved. ing alginate microspheres are compatible with the

Oral vaccination is the preferred route of immuni- use of a variety of antigens (proteins, live bacteriazation to induce mucosal immunity in the gastroin- and viruses) for which immune responses have beentestinal tract, and has several advantages over other demonstrated [4,13,14]. Additionally, alginate micro-mucosal routes. Effectively delivered oral vaccines spheres are stable at low pH [7], and can becan induce mucosal, as well as systemic immunity formulated into a variety of particle sizes that can[1,4]. Additionally, oral immunization uses a physio- pass rapidly through the rumen of cattle [4,15].logical delivery process (ingestion) that is non-inva- Rotavirus infections are one of the major causes ofsive and eliminates needle injections which are diarrhea disease complex in young animals, includ-frequently associated with tissue reactions that cost ing calves and pigs [16,17], and cause significantthe beef industry up to $9 per animal [5]. However, economic loses to the animal health industry [17].successful oral vaccination faces significant chal- Control of rotavirus-induced disease is presentlylenges due to the anatomic and physiologic barriers achieved by passive immunization whereby pregnantpresented by the gastrointestinal tract. Oral adminis- females are immunized during the third trimester oftration of non-replicating vaccine antigens is usually pregnancy and maternal antibodies are transferredassociated with poor immune responses [1,6]. This is from mother to offspring through colostrum and milkin part due to vaccines being degraded in the [16]. While passive immunization has reducedgastrointestinal tract before they reach the gut-associ- rotavirus infection, newborn animals may fail toated lymphoid tissue (GALT), the inductive site for acquire adequate levels of maternal antibodies tomucosal immune responses. Consequently, to achieve effective disease protection. This has led to aachieve effective oral immunization, a ‘protective’ search for approaches to induce active immunity invehicle is required to deliver vaccine antigens to the the intestinal tract of young animals. ProtectionGALT. Many such antigen delivery systems have against rotavirus infection in various species hasbeen evaluated. Biodegradable microspheres have been shown to correlate with intestinal IgA [18,19].proven to be one of the most promising approaches Induction of active immunity in young animals[7,8]. ‘M’ (microfold) cells, located within the would complement the passive immunity fromfollicle-associated epithelium (FAE) overlying the mother’s colostrum or milk. Unfortunately, activePeyer’s patches sample particulate antigens from the immunization of young animals is not extensivelyintestinal lumen. This uptake of particulate material practiced, primarily due to two major concerns: (i)appears to be size dependent. Microparticles with a maternal antibody interference with vaccination [20–diameter less than 10mm are taken up by Peyer’s 22]; and (ii) young animals are thought to have anpatch M-cells [6,9]. Microspheres made from poly- immature immune system [20,21]. These concernslactide-L-glycolide (PLG) have been extensively are supported by the observation that maternalinvestigated with various forms of antigens including antibodies can mediate high level of passive protec-protein and plasmid DNA [6,10]. While PLG micro- tion against rotavirus disease but active immunesphere formulations have successfully induced responses are suppressed in young pigs [23].mucosal immune immune responses, their prepara- There may be effective ways, however, to induce

B. Kim et al. / Journal of Controlled Release 85 (2002) 191–202 193

mucosal immune responses in the presence of mater- insect cell medium (Canadian Life Technologies,nal antibodies. Oral immunization of newborn mice Burlington, ON) supplemented with 10% fetalwith reovirus encapsulated in alginate microspheres bovine serum (Canadian Life Technologies).induced intestinal IgA responses in the presence ofmaternal antibodies while unencapsulated virus did 2 .1.2. Construction of recombinant baculovirusnot [11]. This observation provided evidence that containing rotavirus VP6 gene cDNA insertalginate microspheres could be used to circumvent The rotavirus dsRNA was extracted from purifiedthe suppressive effects of maternal antibodies in virus and denatured by methyl mercury hydroxide.vaccination of young animals. We recently reported The VP6 gene was reverse transcribed into cDNAthat gut-associated lymphoid tissue (GALT) is im- and was amplified by RT–PCR [25,26]. The primers,mune competent in newborn ruminants and that VP6F1 and VP6R1 complimentary to the 59 and 39maternal antibody did not interfere with enteric ends of VP6 gene were synthesized by DNA syn-immunization [24], suggesting that oral immuniza- thesizer (Applied Biosystems) on the basis of thetion may be an effective approach in neonatal sequence of Gottfried (Gene Bank accession numberanimals. Thus, by encapsulating rotavirus vaccine D00326) and restriction enzyme BamH1 site wasantigens in alginate microspheres, it would be pos- introduced into the primers for the convenience ofsible to effectively vaccinate young animals and the gene cloning into the plasmid vector. Theinduce intestinal immunity even in the presence of sequence for the primer VP6F1 wasmaternal antibodies. This approach of a combined

59-CCG GAT CCG GCT TTTimmunization program for mother and offspring ]]]should give maximum disease protection during the AAA CAG AGT CTT C-39 BamH1early days of life when young animals are most

while the sequence for the second primer (VP6R1)susceptible to disease.wasThe objective of this study was to evaluate

whether porcine rotavirus antigens encapsulated in 59-CCG GAT CCG GTC ACA]]]

alginate microspheres could induce mucosal immune TCC TCT CAC TAT A-39 BamH1responses following oral immunization. Since the

The VP6 amplified gene was digested with restrictionoverall goal is to use alginate microspheres inenzyme BamH1 and cloned into the BamH1 site ofruminants, subsequent experiments were conductedthe transfer vector pVL1393 (Invitrogen Co). Thein a sheep intestinal ‘loop’ model to evaluate uptakeconstruct, pVLVP6, placed the VP6 gene under theof microspheres by FAE of Peyer’s patches and tocontrol of the strong AcNPV polyhedrin promoter.determine if uptake of microsphere-encapsulatedRecombinant baculovirus, VP6:175, was constructedprotein induced immune responses in GALT ofby co-transfection of Sf-9 cells with the linearizedruminants.baculovirus DNA and pVLVP6 using a Baculo Goldtransfection Kit (Pharmingen, San Diego, CA). Therecombinant baculovirus was plaque-purified three2 . Methodstimes to eliminate contamination by wild-typebaculovirus [27,28].2 .1. Preparation of VP6 antigen

2 .1.1. Cells and viruses 2 .1.3. Detection and purification of VP6 proteinPorcine rotavirus strain 175 (Group A rotavirus) Baculovirus expressed VP6 was detected by an

was isolated from piglets with diarrhea in South immunofluorescence assay. Sf-9 cells were infectedKorea. Rotavirus 175 was grown in MA-104 cells with recombinant baculovirus VP6:175. After 3–5(provided by Professor Shien-Young Kang, Chun- days, the recombinant baculovirus infected cellsgbuk National University). The recombinant baculo- grown on coverslip were fixed with cold acetone forvirus VP6:175 was propagated in confluent Sf-9 10 min at220 8C.VP6 specific monoclonal antibodymonolayers (ATCC, Manassas,VA) grown in Grace’s or guinea pig hyperimmune 175 rotavirus antiserum


(National Veterinary Research and Quarantine Ser- capsulated in alginate microspeheres by the sprayvice, South Korea) were used to detect the expres- method. Following encapsulation, the micropsheression of VP6. were washed three times to remove free carbon

VP6 protein was partially purified as follows: Sf-9 particles.cells were infected with recombinant baculovirus. Encapsulation of porcine serum albumin (PSA,The infected cells were harvested by centrifugation Sigma) in alginate microspheres was done using a(15003g), resuspended in PBSA and washed two proprietary emulsion-cross-linking technique. Briefl-more times. Cells were lysed by sonication, cen- y, to encapsulate protein, the sodium alginate wastrifuged at 16 0003g and the supernatant was used mixed with PSA and an emulsion was prepared using

as antigen. a high pressure homogenizer (Emulsiflex C5homogenizer, Avestin Inc., Ottawa, CA, USA).

2 .2. Encapsulation of antigens in alginate Alginate droplets were cross-linked with a mixture ofmicrospheres cations and the particle size was measured with a

particle analyzer. The mean volume diameter forThe encapsulation of VP6 protein in alginate microspheres was 9–14mm and greater than 90% of

microspheres was done using the spray method particles had a diameter less than 10mm. Todescribed previously [4], with minor modifications. determine the loading efficiency of PSA, micro-Briefly, VP6 protein or live virus was mixed in a 2% spheres were lysed with 103 PBSA and the superna-solution of medium viscosity sodium alginate tant was then analyzed for total protein using the(Sigma, St. Louis, MO, USA) to a final alginate BCA protein assay (Pierce, Rockford, IL, USA).concentration of 1.2%. The antigen–alginate mixturewas sprayed under 40 p.s.i. of pressure through an2 .3. Immunization of miceultrasonic nozzle (Turbosonic Inc., Waterloo, On-tario, Canada). Alginate droplets were collected into An i.p. immunization experiment was conducteda beaker containing 2% CaCl solution. The alginate to confirm the immunogenicity of the rotavirus2

21cross-links with Ca to form temporary micro- antigens in vivo, and to optimize the VP6-specificspheres, which were further stabilized by incubation ELISA assay. BALB/c mice (Animal Resourceswith 0.2% poly-L-lysine (Sigma). The size of the Center, University of Saskatchewan) were immun-microspheres was determined in a particle analyzer ized with a single i.p. injection with either of the(Model 770 AccuSizer, Particle sizing Systems Inc., following antigens: VP6 suspended in IncompleteSanta Barbara, CA, USA), indicated that 80% of the Freund’s adjuvant (VP6-IFA;n54), VP6 encapsu-microspheres had a diameter of 10mm or less. To lated in alginate microspheres (VP6-MS;n54) ordetermine the efficiency of alginate encapsulation with live porcine rotavirus in incomplete Freund’s(loading efficiency), microspheres containing either adjuvant (PRV-IFA;n54). Control mice were in-the live virus or VP6 protein were lysed by incuba- jected with saline (n52). Serum samples weretion with calcium- and magnesium-free phosphate collected prior to immunization and weekly afterbuffered saline (PBSA, pH 7.2). VP6 antigen was immunization. Mice were killed 4 weeks after im-determined with the Bicinchoninic acid (BCA) pro- munization and mesenteric lymph nodes and Peyer’stein assay (Pierce, Rockford, IL). Virus titer was patch tissues were collected for immune assays.determined by plaque assay with MA-104 cells. For oral immunization experiments, 25 mice wereResults from several experiments consistently allocated equally to five groups and each group wasshowed that the encapsulation efficiency of both housed in a separate microfilter cage (Nalgene,rotavirus protein and virus was approximately 30%. Canada). Mice were orally immunized using aThese loading efficiencies were used to determine gavage needle (CDMV, Quebec, Canada) with a 4the dose of antigen used for immunizations to ensure week interval between each of the three oral immuni-that equivalent amounts of antigen were used for zations. The dose of antigen used was 100mg ofencapsulated and unencapsulated antigen. VP6 protein and 50mg of live PRV virus, either

Colloidal carbon (India ink) was similarly en- encapsulated in alginate microspheres or resuspended


in saline. A final i.p. immunization was done 4 solution was removed by flushing with PBSA beforeweeks after the third oral immunization. Serum was both ends of the ‘intestinal-segment’ were closed.collected on the day of each immunization and The ‘intestinal-segment’ was subdivided into eightweekly after each immunization. Fecal samples were ‘loops’ by tying silk ligatures 20 cm proximal andcollected after the third immunization and weekly distal to each PP to create a space containing anthereafter. To ensure fresh fecal samples were col- individual PP (‘loop’). Interspaces of various lengthslected, mice were moved to clean cages with a separated each ‘loop’ containing a PP. All ‘loops’wire-mesh floor and fecal pellets were collected 30 and interspaces were injected with sodium–ampicil-min later. Mice were euthanized 4 weeks after the lin (Novopharm, Toronto, ON, Canada). PSA antigenfinal immunization at which time intestinal contents encapsulated in alginate microspheres was injectedwere collected from the colon. into individual ‘loops’.

Duplicate ‘loops’ in each animal were injected2 .4. Uptake of microspheres by GALT of sheep with either 0.05, 0.1, 0.5 or 1.0 mg (per ‘loop’) of

PSA encapsulated in alginate microspheres.To examine the interaction between microspheres

and GALT, alginate microspheres were prepared and 2 .6. Organ and cell culturesfiltered through pores of different sizes to yieldmicrospheres with the following approximate diame- 2 .6.1. Miceters: (i) less than 10mm (,10 mm); (ii) greater than Organs from mice were cultured using a procedure10 mm but less than 20mm (10mm,MS.20 mm); described elsewhere [29], with minor modifications.and (iii) greater than 20mm (.20 mm). Micro- Briefly, mesenteric lymph nodes (mesenteric LN)spheres of different sizes were injected into duplicate were removed from the abdomen and washed threeintestinal ‘loops’ containing a jejunal Peyer’s patch times with PBSA containing antibiotics (penincillin,(preparation of intestinal ‘loops’ is described below streptomycin and amphotericin B). Mesenteric LNunder Section 2.5). Intestinal tissue was collected 2 h were divided in half and each portion placed in oneafter injection of microspheres, fixed in formalin and well of a 24-well culture plate containing 1 ml /wellprocessed for routine histology. Tissue sections of serum-free AIM-V medium (GibcoBRL, Burlin-containing both FAE (overlying the PP) and mucosal gton, ON, Canada) supplemented with 2% FBSvillous epithelium were examined with the aid of a (GibcoBRL), 50mM 2-mercaptoethanol (Sigma–Al-light microscope. drich) and antibiotics. Peyer’s patches were dissected

out and similarly processed and cultured in AIM-V2 .5. Immunization of sheep medium. Culture supernatants were collected from

mesenteric LN, and PP after 4 days of culture, andFour month-old suffolk sheep were obtained from stored at220 8C until assayed for VP6-specific

the Department of Animal and Poultry Science, antibody.University of Saskatchewan. Details of anesthesiaand surgery for the preparation of intestinal ‘loops’ 2 .6.2. Sheepwere described previously [13]. Briefly, a midline Lambs were euthanized 3 weeks after immuniza-abdominal incision was made, a segment of jejunum tion in intestinal ‘loop’. PP were collected from eachcontaining eight consecutive jejunal PP was iden- ‘loop’ and two control PP were collected from thetified and transected at both ends (‘intestinal-seg- normal jejunum adjacent to the site of anastomosisment’). Continuity of the jejunum was re-established (outside the ‘intestinal-segment’ with ‘loops’). Thewith an end-to-end anastomosis. The ‘intestinal-seg- PP were dissected and placed separately in 10 mlment’ was flushed with PBSA to remove ingesta and ice-cold PBSA containing 0.1% ethylenediamine-an antibiotic solution (metronidazole, Abbot Lab- tetraacetic acid (EDTA; BDH Inc., Toronto, ON,oratories, St. Laurent, PQ, Canada; enrofloxazine, Canada). Intestinal contents were collected by cuttingBayer Inc., Etobicoke, ON, Canada) was infused each ‘loop’ open along the mesenteric border andthroughout the ‘intestinal-segment’. The antibiotic contents were added to PBSA at a 1:5 w/v ratio, and


stored at270 8C. To assay for secreted antibody, supernatants and incubated at room temparature forcontents were thawed, particulate material was re- 15 min. Fetal bovine serum was added and themoved by centrifugation at 6003g for 15 min and samples were store at220 8C.the supernatant used for immune assays. The PP VP6-specific IgA in fecal samples was determinedcells were isolated as previously described [30]. by ELISA. Microtiter plates were coated with VP6Briefly, each PP was rinsed with ice-cold PBSA and protein and 100ml of processed fecal samples wereplaced in cold PBSA containing 0.1% EDTA. added to each well. Wells were washed and thenLymphoid follicles and cells from the interfollicular incubated first with biotinylated goat anti-mouse IgAregion were released by mechanically separating the (Cedarlane Laboratories, Hornby, ON) and then withmucosa from the underlying muscularis externa and alkaline phosphatase-conjugated streptavidin (Jack-mucosal fragments were removed before preparing a son ImmunoResearch Lab., Inc., Westgrove, PA).single-cell suspension by repeatedly pipetting to Alkaline phosphatase activity was detected withdisrupt lymphoid follicles. The resulting cell suspen- PNPP substrate (Sigma) and absorbance was read assion was filtered through a 40mm nylon mesh (Cell described above and optical density (O.D.) wasStrainer; Becton Dickinson Labware, NJ, USA), reported.washed once in PBSA and resuspendend in culturemedium. Viable cells were identified by trypan blue

2 .7.2. Detection of PSA-specific antibody secretingexclusion and counted with a haemocytometer.

cells and secreted antibody in sheep intestinePSA-specific antibody secreting cells (ASC) were

2 .7. Antigen specific immune assaysdetected using a modified ELISPOT assay describedpreviously [13]. Briefly, microtiter nitrocellulose

2 .7.1. Detection of VP6 specific antibodies in micefiltration plates (Whatman Inc., Clifton, NJ, USA)

VP6-specific serum IgG was detected as follows: 6were coated with PSA (500mg/ml) and 0.1310 PP96 well microtiter plates (DYNEX Technologies, 6cells /well or 0.5310 PP cells /well were added toInc., Chantilly, VA) were coated overnight with 0.5

triplicate wells. Following overnight culture, PSA-mg per well of partially purified VP6 protein. The

specific ASC were detected by first addingplates were washed with PBS containing 0.05%

biotinylated rabbit anti-sheep IgG (H1L chain spe-tween 20 (PBST). Serum samples were diluted 1:100

cific; 1:6000 dilution; Kirkegaard and Perry Lab-and added to wells. VP6-specific IgG or IgA was

oratories, Gaithersburg, MD, USA), followed by AP-detected with alkaline phosphatase-conjugated goat

conjugated streptavidin (1:1000 dilution; Jacksonanti-mouse IgG or IgA (Kirkegaard and Perry Lab-

Immunoresearch, Lab. Inc., Westgrove, PA, USA).oratories, Inc., Gaithersburg, MD), respectively. The

ASC were visualised with 5-bromo-4-chloro-3-in-color reaction was developed withp-nitrophenyl

dolyl phosphate/nitroblue tetrazolium (BCIP/NBT)phosphate (PNPP) substrate (Sigma, St. Louis, MO).

insoluble alkaline phosphatase substrate (Sigma–Al-Absorbance was read at 405 nm wavelength on a

drich). The frequency of PSA-specific ASC per 13BIO-RAD model 3550 microplate reader (BIO-RAD 610 cells was calculated by subtracting the numberLaboratories, Hercules, CA), and reported as optical

of spots present in wells not coated with PSA fromdensity (O.D.) above that of a negative control

the number of spots present in PSA-coated wells. Ansample.

inverted light microscope was used to count fourFeces and intestinal contents were treated with

quadruplicate wells for each cell population and dataprotease inhibitors [31] to prevent degradation of

presented are mean values.immunoglobulins by proteases before anti-VP6 anti-bodies were assayed. Samples were dissolved in coldPBSA at a ratio of 1:32 (w/v), vortexed vigorously, 2 .8. Statistical analysisand any clumps were dispersed with a pipette.Samples were centrifuged, the pellet was discarded Data (except pooled fecal samples) were analyzedand the supernatant was further clarified by centrifu- by a Kruskal–Wallis one-way nonparametric analysisgation. PMSF and sodium azide were added to of variance using Statistix software (Analytical Soft-


ware, Tallahassee, FL). The level of significance wasset atP50.05.

3 . Results

3 .1. Mouse studies

An i.p. immunization experiment was conductedto confirm the immunogenicity of the recombinantrotavirus VP6 protein in vivo. VP6-specific IgGantibody responses were detected in sera of mice 4weeks after a single i.p. immunization with VP6-IFA,VP6-MS and PRV-IFA, and antibody levels in thelatter group were significantly elevated above salinecontrols (Fig. 1a). No VP6-specific IgA was detectedin serum (data not shown). In contrast, VP6-specificIgA antibodies were detected in culture supernatantsof mesenteric lymph nodes from mice immunizedi.p. with VP6-IFA and PRV-IFA but not with VP6-MS (Fig. 1b). No VP6-specific IgG was detected inculture supernatants of mesenteric LN (data notshown) and no VP6-specific antibodies of either IgGor IgA isotype were detected in culture supernatantsof PP (data not shown). These observations con-firmed the immunogenicity of the VP6 vaccineantigen and indicated that vaccine formulation couldchange the immunogenicity of the VP6 protein.

After confirming the immunogenicity of therotavirus antigens, we then evaluated whether oralimmunization with rotavirus antigens formulated inalginate microspheres would induce systemic andmusosal immune responses. Antibody responseswere then assayed in sera of orally immunized mice.Three oral immunizations induced elevated VP6-specific IgA antibody responses in pooled fecalsamples from all groups of orally immunized micecompared to controls (Fig. 2a). However, no fecalIgG was detected (data not shown). Oral immuniza-tion did not induce any detectable serum VP6-spe-cific antibodies (IgG or IgA) (data not shown). Whenorally immunized mice and the saline controls wereinjected i.p. with VP6-IFA, the i.p. immunization

Fig. 1. VP6-specific antibody responses in (a) serum, and in (b)boosted mucosal immune responses as indicated byculture supernatants of mesenteric lymph nodes of mice 4 weeksthe higher IgA antibodies detected in feces collectedafter a single i.p. immunization with VP6 protein in incomplete

from mice 10 days after the i.p. injection (Fig. 2b). Freund’s adjuvant (VP6-IFA),VP6 in alginate microspheres (VP6-Modest fecal IgA was detected in mice after the MS), PRV in incomplete Freund’s adjuvant (PRV-IFA) or salineprimary i.p. immunization of the control group, alone. Data presented are mean6S.E.M. of values for each group.


confirming our earlier observation that a single i.p.immunization can induce an intestinal IgA response.

Results from the pooled fecal samples were con-firmed when intestinal contents collected from in-dividual mice 18 days after the i.p. injection wereassayed for antibody responses. VP6-specific IgAantibody responses were detected in the intestinalcontents of mice in all groups (Fig. 3). The lowerIgA responses detected in intestinal contents fromindividual mice (Fig. 3) compared to pooled fecalsamples (Fig. 2b) may be due the fact that the formersamples were collected 8 days after that the latterwhen IgA levels may have been declining. TheVP6-MS group had the highest IgA antibody re-sponse, but was not significantly different from non-encapsulated VP6.

3 .2. Sheep studies

The efficacy of microsphere formulations isthought to be due to the increased uptake by M cells.Therefore, the interaction between alginate micro-spheres and FAE was investigated. Microspherescontaining colloidal carbon were attached only to theFAE, the epithelium overlying the PP follicles, but

Fig. 2. VP6-specific IgA in feces of mice after (a) three oralimmunizations and (b) after three oral followed by an i.p.immunization. Mice were orally immunized three times with Fig. 3. VP6-specific IgA in intestinal contents of mice after threeeither VP6 in alginate microspheres (VP6-MS), VP6 protein oral followed by one i.p. immunization. Mice were orally immun-(VP6), porcine rotavirus (PRV) porcine rotavirus in alginate ized three times with either free VP6 in alginate microspheresmicrospheres (PRV-MS), PRV in saline or saline alone (control). (VP6-MS), VP6 (VP6), porcine rotavirus in alginate microspheresAn i.p. booster immunization was done with VP6 protein in IFA. (PRV-MS), PRV in saline or saline alone (control). i.p. boosterData presented are O.D. for pooled fecal samples (one sample per immunization was done with VP6 protein in IFA. Data presentedgroup, hence no error bars). Dotted line indicates background O.D. are mean6S.E.M. of values for each group. Dotted line indicatesfrom fecal samples of non-immunized control mice (Fig. 2a) background O.D. from fecal samples of non-immunized controlanalyzed within the same assay. mice (Fig. 2a) analyzed within the same assay.


were not attached to the villous epithelium in the not in those from ‘loops’ injected with larger micro-non-Peyer’s patch areas (Fig. 4). Additionally, spheres (10–20mm and .20 mm). Furthermore,microspheres attached to the FAE only in sections following a 2 h period, colloidal carbon was visiblefrom loops injected with microspheres,10 mm but within the lymphoid follicles of the PP (Fig. 4). The

Fig. 4. Uptake of alginate microspheres by GALT of ruminants. Colloidal carbon was encapsulated in alginate microspheres and injectedinto intestinal ‘loops’ of sheep containing jejunal PP. Tissue sections were examined under a light microscope. Alginate microspherescontaining colloidal carbon (arrows) were seen attached to the follicle-associated epithelium and in follicles of jejunal Peyer’s patches.


the intestinal tract following oral immunization ofmice. Alginate-encapsulated recombinant proteinappeared to induce a slightly higher intestinal IgAresponse compared to unencapsulated VP6 protein.The uptake of microspheres in the intestinal tract is asize dependent phenomenon. Microparticles with adiameter of less than 10mm were taken up by ‘M’cells in the Peyer’s patches of mice and pigs,respectively [6,9]. The present investigation confirmsthat in the GALT of ruminants, as in the otherspecies, microspheres,10 mm are taken up by FAEwithin which ‘M’ cells are contained. Most im-portantly, colloidal carbon encapsulated in alginatemicrospheres was visualized in the follicles of PPwhere induction of immune responses occurs. Theseobservations indicate that alginate microspheres‘target’ PP (and presumably ‘M’ cells) and deliverencapsulated material to the intestinal immune sys-tem. In subsequent experiments we demonstratedthat the uptake of microspheres was followed byFig. 5. Frequency of PSA-specific antibody secreting cells (IgG

and IgA) in Peyer’s patches of sheep. Intestinal ‘loops’, each successful induction of immune responses as indi-containing a jejunal PP were injected with PSA encapsulated in cated by the detection of PSA-specific antibodyalginate microspheres. Immune responses were assayed by ELIS-

secreting cells in PP.POT assay 3 weeks after a single immunization and data presentedThe pattern of redistribution of microspheres isare means (6S.E.M.) of ASC counted from four ‘loops’.

also dependent on their size. Eldridge and coworkers[6] showed that microspheres within the range of5–10 mm were taken up by Peyer’s patches and

uptake of microspheres was also confirmed by the remained in the dome area, while those,5 mm werepresence of numerous PSA-specific antibody secret- detected in the Peyer’s patches, mesenteric lymphing cells (ASC) of IgG and IgA (Fig. 5) isotypes nodes and spleen. This suggests that particle sizecollected from ‘loops’ injected with PSA-MS. The may determine the type of the immune responsenumber of ASC within the PP also correlated with elicited by vaccine-containing microspheres adminis-the dose of encapsulated antigen injected into each tered by the oral route. In this regard, microspheresintestinal ‘loop’. No ASC cells were detected in less than 5mm would be predicted to induce acontrol PP collected from the normal adjacent je- predominantly circulating antibody response basedjunum (jejunum outside the ‘intestinal-segment’ with on their propensity to disseminate to systemicthe ‘loops’) of animals with immunized intestinal lymphoid tissue. In contrast, microspheres greater‘loops’. than 5mm would be predicted to raise predominantly

a mucosal immune response because they remain inthe IgA inductive environment of the Peyer’s patches

4 . Discussion over the course of antigen release [6]. Thus, based onthe size characteristics of the microspheres made

The encapsulation of antigens in microspheres is using our procedure, we could speculate that therethought to reduce antigen degradation and enhance were relatively more microspheres in the 5–10mmantigen delivery to GALT [1,7]. The present in- diameter range, or microspheres of this size con-vestigation confirmed that encapsulating PRV and its tained a sufficient amount of VP6 antigen to induce aVP6 protein in alginate microspheres effectively detectable mucosal IgA response.induced antigen specific IgA antibody responses in i.p. immunization induced a primary intestinal IgA


response in the group of mice that had not been the intestinal tract following oral and enteric ad-previously immunized orally (‘control’). Additional- ministration.ly, the i.p. immunization enhanced enteric IgAresponses induced by prior oral vaccination. Recentreports have confirmed that the mesenteric lymph A cknowledgementsnodes are an alternate site for the induction ofintestinal IgA responses in the absence of Peyer’s We thank Drs D. Godson, M. Baca-Estrada forpatches [32]. In this regard, it is likely that i.p. their advice, Dr H. Townsend for help with theinjection of native protein or PRV drains to the statistical analysis and Dr D. Wilson for help with themesenteric lymph node, thereby inducing IgA re- surgery model. We also thank B. Carrol, Donna Dent,sponses in the mesenteric LN. Effector lymphocytes and C. Olson for their technical assistance. Financialwould then disseminate to the intestinal tract as support for this work was provided by Saskatchewanindicated by the detection of IgA in intestinal HSURC, Natural Sciences and Engineering Researchcontents. No IgA antibodies were detected in culture Council and Western CARDS. Dr L.A. Babiuk is asupernatants of PP mice immunized i.p. with the holder of the Canada Research chair in vaccinology.rotavirus antigens. This is not surprising since effec- Published with permission of the director of VIDOtor cells such as IgA plasma cells disseminate as VIDO journal series no. 311.primarily to the lamina propria from where theysecrete the IgA. The IgA in turn is transported acrossthe intestinal wall into the lumen by a receptor- R eferencesmediated process. However, when the antigen isgiven orally, it is taken up from the lumen across the [1] W. Muir, A.J. Husband, E.M. Gipps, M.P. Bradley, Inductionintestinal wall into the Peyer’s patches, where induc- of specific IgA responses in rats after oral vaccination with

biodegradable microspheres containing a recombinant pro-tion of immune responses occurs, leading to gene-tein, Immunol. Lett. 42 (3) (1994) 203–207.ration of effector cells and the presence of IgA in

[2] A.M. Mowat, J.L. Viney, The anatomical basis of intestinalfeces. The booster effect of i.p. immunization in immunity, Immunol. Rev. 156 (1997) 145–166.mice previously immunized orally suggests cross- [3] Z.Q. Xiang, S. Pasquini, H.C. Ertl, Induction of genitalcommunication between systemic and mucosal im- immunity by DNA priming and intranasal booster immuniza-

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was recovered from lysed microspheres (unpublished [6] J.H. Eldrige, C.J. Hammond, J.A. Meulbroek, J.K. Staas,observation). We speculate that encapsulation may R.M. Gilley, T.R. Tice, Controlled release in the guthave prevented the interaction between PRV and associated lymphoid tissue. I. Orally administered biodegra-

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