Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge

PII: SO264-410X(98)00085-1

Vaccme, Vol. 16, No. 20, pp. 2010-2017. 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/98 $19+0.00

ELSEVIER

Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge Philippa L. Heritage *, Brian J. Underdown*, Mark R. McDermott*

Michael A. Brook? and

The mucosal and systemic humor-al immune systems can function essentially independent of each other; responding to mucosal and parenteral antigens, respectively. Nevertheless, antigen administered by one route can modi& responsiveness to subsequent immunization by an alternate route. Here we demonstrated, in mice, in addition to stimulating rapid and robust sera antibody responses, intragasttic (i.g.) immunization with human serum albumin (HSA)-containing starch microparticles (h4P) grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS) primed for enhanced specific sera IgG following a parenteral antigen boost. After as little as one i.g. immunization with microentrapped, but not with soluble, HSA antigen-specific proliferation and antibody secretion were detected in Peyer’s patches (PP); this activity peaked after three i.g. MP immunizations. We observed a progressive dissemination of antigen-specific lymphocyte reactivity j?-om PP to splenic tissue following oral MP immunization. Similarly, we observed a shift in HSA-specific antibody-secreting cells from PP and mesenten’c lymph nodes to splenic tissue following i.g. MP immunization. We also demonstrated that oral immunization with microentrapped, but not with soluble HSA, resulted in enhanced numbers of spontaneous Th2-cytokine secreting lymphocytes which disseminated from mucosal to systemic lymphoid compartments. This observation coincided with our findings that HSA-specific sera IgGl responses in animals given HSA in MP were significantly higher than those detected in the sera of mice given soluble HSA i.g., both before and after parenteral antigen challenge. These findings suggest that orally-administered TS-PDMS-grafted MC: by stimulating elements of the mucosal immune system, are a valuable addition to mucosal and systemic vaccine delivery systems. 0 1998 Elsevier Science Ltd. All rights reserved Keywords:

Microparticles;

vaccine; mucosal immunity

Presently, most vaccines are administered via systemic routes and although parenteral immunization is effective in stimulating systemic immunity, specific mucosal immunity is not often induced. In contrast, mucosal immunization provokes both mucosal and systemic immunity via the stimulation of mucosallysituated IgA and IgG plasmacyte precursors’,‘. These observations predict that mucosal, but not parenteral, immunization induces immune responses which are Departments of *Pathology and Chemistry and tchemistry, McMaster University, Hamilton, Ont. L8N 325, Canada. *Author to whom correspondence should be addressed. Tel: (905) 525-9140, ext. 22873; fax: (905) 570-0742; e-mail: [email protected] (Received 20 November 1997; revised version received 9 February 1998; accepted 6 March 1998)

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capable of both protecting the host from mucosal invasion and eliminating intruders from systemic tissues if the mucosae are breached. In concert, these mucosae-derived responses prevent or reduce the morbidity and mortality resulting from both mucosal and systemic infections3. We previously demonstrated that human serum albumin (HSA)-containing, biocompatible starch microparticles (MP) grafted with the silicone polymer 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS) stimulated both secretory and circulating antigen-specific humoral immunity following intragastric (i.g.) immunization4. Maximal sera HSA-specific responses occurred following oral boosting4. This response was manifested in the systemic compartment of the immune system and suggested that

Priming of GALT with oraf microparticles: P.L. Heritage et the initial immunization protocol could induce systemic humoral memory. Indeed, some reports suggest that feeding soluble antigens can sensitize animals for systemic DTH responses”-‘. Additionally, since orallyinduced memory cells have been repeatedly demonstrated to be activated following subsequent antigen challenge with cholera toxoid or its subunitsXm”, a potent mucosal and systemic antigen, i.g. priming of mucosal tissues with TS-PDMS-grafted MP might also lead to the generation of systemic memory capability, a phenomena not consistenl:ly seen with other particulate delivery system?. In this study, we investigated whether orally-administered HSA-containing TS-PDMS-grafted MP primed mice for a systemic challenge with soluble antigen. The results showed that i.g. immunization with low doses of microentrapped, but not soluble. HSA activated lymphocytes in the gut-associated lymphoid tissue (GALT) and primed animals for significantly enhanced HSA-specific humoral immune responses following intraperitoneal (i.p.) HSA challenge. The results further support the use of TS-PDMS-grafted MP as a delivery vehicle for mucosal and systemic vaccines. MATERIALS AND METHODS Microparticle

fabrication

and characterization

HSA (Sigma, St. Louis, MO) was entrapped into TS-PDMS-grafted MP usmg an emulsion-based process and characterized as previously described”. Microparticles fabricated by this process routinely contained 5-6% wlw protein. Immunizations

Female BALBic mice, age 6-8 weeks (Harlen Sprague Dawley Inc.. Indianapolis, IN) were immunized i.g. on days 0, 7 and 14 with 5Opg of soluble or microentrapped WA in 500 ~11of 0.2 M NaHC03, or vehicle alor’e using PE50 tubing (Becton Dickinson Co., Mountain View, CA). In some experiments, animals were subsequently immunized intraperitoneally (i.p.) on day 19 with 100 pg HSA solubilized in phosphate-buffered saline (PBS, pH 7.4). Collection

and preparation

of sera and cells

Individual blood samples were obtained via the retro-orbital plexus. Fo:r sera evaluations, insoluble material was removed by centrifugation and sera were stored at - 70°C until used. Pooled spleens (SPL), mesenteric lymph nodes (MLN) and PP were isolated into ice cold Hanks’ balanced salt solution (HBSS, pH 7.4). Single cell suspensions of SPL and MLN were prepared by crushing the tissues between the frosted ends of two microscope slides. Cell suspensions of PP were prepared as previously described4. Single cell SPL, MLN and PP suspensions were washed twice with HBSS by centrifugation, and the erythrocytes and dead cells were removed using Ficoll-Paque” (Pharmacia, Uppsala, Sweden). The remaining cells were resuspended in RPMI-1640 media supplemented with 5% heat-inactivated fetal bovine serum, 1% penicillinstreptomycin, 1% L-glutamine and 1% N-n-hydroxyethylpiperizine-N’-2 ethanesulfonic acid (HEPES), pH

al.

7.4 (operationally termed RPMI-5). The viability of cell preparations routinely exceeded 90% as judged by ethidium bromideifluorescein diacetate staining’ .14. Measurement

of HSA-specific

antibody responses

An enzyme-linked immunosorbant assay (ELISA) was used to measure HSA-specific antibody responses in individual sera as previously described4. Briefly, duplicate serial dilutions of sera were incubated on HSA-coated microtitre plate wells. HSA-specific antibodies were quantitated by incubating wells with heavy chain-specific, alkaline phosphatase-conjugated goat anti-mouse IgG, IgGl or IgG2a (Southern Associates, Birmingham, AL). Biotechnology Responses were quantitated by measurement of optical densities (OD) at 405 nm following incubation of wells with 1.0 M diethanolamine buffer, pH 9.8, containing 50 mM MgCl, and 1.0 mg ml-’ p-nitrophenylphosphate (5 mg phosphatase substrate tablets; Sigma). Sera prepared from buffer-treated animals were used to establish baseline OD values. The results were expressed as reciprocal end-point sera titres representing the greatest sera dilutions giving OD values exceeding two times buffer-alone mean values. Lymphocyte

proliferative

assay

Quadruplicate cultures of PP, MLN and SPL lymphocytes (5 x 10’) were prepared in 96-well, roundbottomed, sterile plates in the presence or absence of various amounts of HSA (250-1000 pg ml-‘) for 96 h at 37°C with 5% CO1 in air. One &i of [3H]thymidine ([‘H]Tdr, 740.0 GBq mmolK ‘; DupontiNEN, Mississauga, ON) was added to each well for the final 24 h of culture. Cells were harvested with a PhD Harvester (Cambridge, MA) and [“H]Tdr incorporation was measured by standard liquid scintillation counting methods. The results were expressed as mean cpm from quadruplicate cultures. Enumerating

of HSA-specific

spot-forming

cells

An enzyme-linked spot-forming assay (ELISPOT) was used to detect HSA-specific spot-forming cells (SFCs) in pooled PP, MLN and SPL preparations as previously described4. Duplicate serial dilutions of single cell suspensions (beginning at 1 x lo5 per 100 ~1 per well) were examined using nitrocellulose plates (Millititer HA; Millipore Corp., Bedford, MA) incubated with 100 /d HSA (100 pg ml-’ in PBS). The results represent the mean number of spots per 1 x 10h cells in duplicate wells containing at least twofold more spots than wells incubated with cells from buffertreated animals. Enumeration

of IFN-7 and IL-4-producing

cells

An ELISPOT assay was also used to quantitate spontaneous cytokine spontaneously released by MLN and SPL cells in vitro. Spontaneous cytokine secretion was examined since we concluded that by examining cytokine production immediately following lymphocyte isolation, and not after extensive in vitro manipulation, we would have a closer approximation of the in vivo cytokine microenvironment following i.g. immunization with microentrapped or soluble antigen. To detect

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interferon (IFN-)y-secreting cells, duplicate serial dilutions of single cell suspensions (beginning at 1 x lo5 cells per 100 ~1 per well) were examined using nitrocellulose microtitre plates (Millititer HA; Millipore Corp) previously incubated overnight at 4°C with 100 ~41per well of a rat anti-mouse IFN-y monoclonal antibody (mAb) (clone R46A2; Pharmingen; SanDiego, CA) at 10 pgmll’ in a solution of 0.1 M NaHC03, pH 8.2 (coating buffer) and then washed with PBS and treated with 250 ~11per well of 0.1% gelatin in PBS. After an 8 h incubation at 37°C the wells were washed three times each with PBS and PBS containing 0.05% Tween-20 (PBSTween). The wells were then incubated for 2 h with 100 ~11of biotinylated IFN-)I mAb (clone XMG1.2; rat anti-mouse Pharmingen) diluted to 4 /lg mlm PBS/Tween containing 0.1% gelatin (dilution buffer). The wells were washed three times each with PBS and PBS/Tween and incubated for 30 min at 37°C with alkaline phosphatase-conjugated streptavidin (Southern Biotechnology Associates) in dilution buffer. After washing the wells four times each with PBS and spots were visualized as previously PBSlTween, described’. The results represent the mean number of spots per 1 x lo6 cells in duplicate wells containing at least twofold more spots than wells incubated with cells from PBS-treated mice. To detect IL-4-specific SFCs, rat anti-mouse IL-4 (clone IlBll; Pharmingen) (2 ,Llgml-‘) and biotinylated mAb anti-mouse IL-4 (clone BVD6-24G2; Pharmingen) (2 /lg ml-‘) were used in coating and detection, respectively. IFN-:I and IL-4 secretion was examined since these cytokines are usually produced by helper T (Th) 1 cells and Th2 cells, respectively, and reciprocally antagonize the effector function of the other Th subset. Statistical

analyses

Statistical analyses were done using GraphPad Prizm’ Version 2.00 (GraphPad Software, San Diego, CA). One-way ANOVA, Tukey test pair-wise multiple comparisons and unpaired Student’s t-tests were used to detect and compare mean differences between treatment groups. A level of significance of 95% was chosen for all tests.

enhanced specific sera antibody responses after systemic challenge at an earlier time with HSA. Figure IB shows that following i.p. boosting with soluble HSA, mice previously given microentrapped HSA i.g. elicited higher levels of anti-HSA sera IgG as compared to those given soluble antigen orally @<0.05) or buffer alone 0,
l(4

zoooj T

Prechallenge

RESULTS Intragastric administration primes mice for subsequent

of microentrapped systemic challenge

HSA

We previously showed that i.g. immunization with low doses of HSA encapsulated in TS-PDMS-grafted MP induced antigen-specific sera IgG titres which were significantly higher than those elicited by comparable amounts of soluble HSA3. Figure IA confirms these findings by showing that i.g. delivery of low doses of microentrapped HSA (50 big per mouse) rapidly elicited levels of specific sera IgG which were greater than those measured in animals given equivalent doses of soluble antigen orally (p < 0.005). Our earlier work4 demonstrated also that following i.g. immunization with HSA-containing TS-PDMS grafted MP, maximal serum antibody titres occurred after oral boosting, on day 70, with microentrapped HSA. Thus, we explored whether i.g. immunization with microentrapped HSA could also prime mice for

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Post Challenge Figure 1 Sera anti-HSA IgG antibody responses. Groups of 10 mice each were immunized i.g. on days 0, 7 and 14 with 50 1/g of HSA incorporated in TS-PDMS-grafted microparticles (MP, solid bars), HSA in 0.2 M NaHCO, (hatched bars) or vehicle alone (open bars). On day 19, animals were boosted i.p. with 100 j(g of HSA solubilized in phosphate-buffered saline. Sera obtained on day 15 (a) and day 33 (b) were evaluated for the presence of HSA-specific IgG using an ELISA. Results are expressed as mean reciprocal end-point titres representing the greatest serum dilutions giving OD values exceeding twice the buffer-treated mean values + SEM.

Priming of GALT with oral micropatticles:

robust humoral immune responses (F&-e IA) and also primed mice for enhanced sera IgG responses following systemic antigen challenge. Characterization of HSA-specific proliferative responses

lymphocyte

Early studies indicated that oral immunization with soluble and particulate antigens can elicit systemic antibody responses due to the dissemination of cells from the GALT15.16. Since particulate antigens are selectively taken up from the intestinal lumen by PP following i.g. immunizauon”-“, we explored whether i.g. administration of TS-PDMS-grafted MP might prime mice for enhanced systemic humoral responses following parenteral antigen challenge via activation and subsequent emigration of primed PP cells. Within 4 days after i.g. administration of MP, antigen-specific PP cell proliferation was detectable following in vitro restimulation with a high dose (1000 fig mll’) of HSA (Table I). This response rose to a maximum by day 16 post-immunization as judged by the efficacy of lower in vitro restimulatory doses of HSA and higher levels of “[H]Tdr incorporation. No significant SPL cell proliferation was detected following in vitro HSA restimulation until 10 days after i.g. immunization with HSA-containing MP, although substantial proliferation was measured at day 16. Moreover, no significant PP or SPL proliferation was observed in mice immunized i.g. with soluble HSA or buffer alone (data not shown). These results indicated that a single i.g. immunization with microentrapped, but not soluble, HSA resulted in the induction of antigen-specific PP cells and that multiple i.g. MP admimstrations resulted in a shift of HSA-specific lymphocyte responsiveness from the mucosal (PP) towards the systemic (SPL) compartment of the immune system. Kinetics of HSA-specific B cell responses following intragastric immunization with microentrapped HSA

Intragastric immunization with HSA-containing TS-PDMS-grafted MP activated PP lymphocytes

P.L. Heritage et al.

(TabZe 1) and elicited antigen-specific serum IgG (Figure IA), suggesting that antigen-specific B cells were stimulated initially in the GALT. Thus, we secretion by HSA-specific antibody examined gut-associated and peripheral lymphocytes at various times after i.g. immunization with encapsulated or soluble HSA (Figure 2). Following primary i.g. immunization with HSA-containing TS-PDMS-grafted HSA-specific numbers of appreciable MP, IgM-secreting SFCs were detected only in PP cell isolates (Figure 2A). The numbers of IgM-secreting cells in PP and MLN (Figure 2B) increased substantially until day 21 post-immunization, after which time no specific IgM SFCs were detectable in either tissue. HSA-specific IgM-secreting SFCs were undetectable in SPL cell isolates until 3 weeks post-immunization (Figure 2C). As certain types of MP have been shown to accumulate primarily in the GALT following i.g. administration”, these results suggest that i.g. administration of microentrapped HSA activated antigenspecific B cells in PP and these, or MP-containing PP-derived phagocytes, subsequently migrated to the SPL via the MLN’3.‘4. Antigen-specific IgG-secreting SFCs in PP and MLN (Figure 2D and 2E, respectively) were not detected until day 21, thus implying that isotype switching of activated, HSA-specific B cells had occurred by this time. The greatest numbers of IgG-secreting SFCs in PP and MLN appeared 3 weeks after primary immunization (Figure 2D and 2E, respectively) while maximal splenic IgG production was not observed until day 28 (Figure 2F), which suggested that IgG plasmacyte precursors might have migrated also from PP through MLN. In contrast, no appreciable numbers of IgM- or IgG-secreting SFCs were observed in the GALT following i.g. immunization with soluble HSA; this confirmed our previous finding that soluble HSA failed to activate PP lymphocytes (Table 1). These results demonstrate that orally delivered, microentrapped HSA activated gut-associated B cells; such activation with microentrapped, but not soluble, HSA might be responsible for priming GALT-derived cells for a recall response to i.p. challenge with antigen.

Table 1 Proliferative responses in Peyer’s patches (PP) and spleens (SPL) following intragastric immunization

with human serum albumin

(HSA) 3H-thymidine

incorporation

(cpm)

HSA (pg ml -‘)

lnoculant TS-PDMS-grafted

MP

Cell source

in vitro

Day 4

Day 10

Day 16

PP

1000 500 250 0 1000 500 250 0 1000 500 250 0 1000 500 250 0

646_+81 195+11 184&9 164k9 186+20 191*11 227 & 29 192k16 187k13 240+51 214k15 186k15 206klO 199*27 279 f 47 208 & 65

1976 & 448 1003f69 397&118 181 kll 873+127 603k104 1795*17 215k13 596 + 45 331*29 317*35 247 f 20 307 & 52 207+9 285+21 315&45

2090 i 446 1760 _t 347 514*41 290 ) 49 4282 i 1203 1927 + 406 829 f 44 195*17 5OOi16 357 f 43 176k4 186i29 225 * 32 231 k17 186k9 213*9

SPL

Soluble HSA

PP

SPL

Groups of 10 mice were immunized intragastrically on days 0, 7 and 14 with 50 pg of HSA incorporated into TS-PDMS-grafted microparticles (MP) or HSA in 0.2 M NaHCO,, On day 4, 10 or 16, animals were sacrificed and lymphocytes isolated from PP or SPL were examined for ‘Hthymidine (‘H-Tdr) incorporation following in vitro incubation for 96 h with or without HSA (250-1000 pg ml-‘) with ‘H-Tdr added to cultures in the final 24 h. Results are expressed as the mean cpm of quadruplicate cultures +SEM.

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Lymphocyte cytokine production following intragastric immunization with microentrapped HSA

We previously showed that HSA-specific sera antibody responses induced after i.g. immunization with HSA-containing TS-PDMS-grafted MP were predominantly IgG14. Since isotype directly reflects the cytokine microenvironment acting on B cells, we examined spontaneous (non-restimulated) cytokine by mucosally- and systemically-derived secretion lymphocytes following i.g. immunization with soluble or microentrapped HSA (Table 2). When compared to mice immunized i.g. with soluble HSA, those given MP i.g. contained appreciable numbers of IL-4-secreting lymphocytes in the PP 2 weeks after primary Kg. immunization; later, IL-4 secretion declined in PP and rose in SPL cells. Conversely, spontaneously secreting IFN-7 cells were rarely observed in PP or SPL following i.g. immunization with either soluble or microentrapped HSA. However, both IFN-~8 and IL-4 secreting cells were detected following stimulation with Con A for 3 days in vitro (data not shown), confirming that the ELISPOTS were working and the multipotent cytokine potential of MALT-derived lymphocytes.

4

0

(a)PP

(4PP

I

I

14

21

L

Day Post Immunization Figure 2 Appearance of HSA-specific spot-forming cells (SFCs) in Peyer’s patches (PP), mesenteric lymph nodes (MLN) and spleens (SPL). Groups of 10 mice each were immunized i.g. on days 0, 7 and 14 with 50 pg of HSA entrapped in TS-PDMS-grafted MP (solid bars), HSA in 0.2 M NaHCO, (hatched bars) or buffer alone. On days 7, 14, 21 and 28, animals were sacrificed and lymphocytes isolated from PP (A, D), MLN (8, E) and SP (C, F) were evaluated for the presence of HSA-specific IgM (A-C) and IgG (D-F) SFCs using an ELISPOT. Results represent the mean number of spots per 1 x lo6 pooled cells in duplicate wells containing at least twofold more spots than wells incubated with pooled cells from buffer-treated animals.

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Table 2 Detection of spontaneously-secreting IFN-; and IL-4 spot-forming cells (SFC) in Peyer’s patches (PP) and spleens (SPL) following intragastric immunization with soluble or microentrapped HSA Cytokine-specific SFCs/l O6 cells lnoculant TS-PDMS-grafted MP

Cell source PP

SPL

Soluble HSA

PP

SPL

Day

IFN-;

IL-4

7 14 21 7 14 21 7 14 21 7 14 21

ND 5 5 ND ND ND ND ND ND ND 10 ND

ND 360 280 ND 30 150 ND 10 5 ND ND ND

Groups of 10 BALB/c mice each were immunized Lg. on days 0, 7 and 14 with 50 pg of HSA incorporated into TS-PDMS-grafted MP, HSA in 0.2 M NaHCO, or buffer alone. On days 7, 14 and 21 animals were sacrificed and lymphocytes isolated from Peyer’s patches (PP) and spleen (SPL) were evaluated for IFN-y or IL-4 secretion using an ELISPOT. The results shown represent one of two experiments and demonstrate the mean number of spots per 1 x lo6 pooled cells in duplicate wells containing at least twofold more spots than wells incubated with pooled cells from buffertreated animals. ND, not detectable.

with Thus, i.g. immunization HSA-containing TS-PDMS-grafted MP incited higher numbers of gut-associated lymphocytes to secrete cytokines known to support IgGl synthesis. Concomitant with these observations, HSA-specific IgGl sera responses in animals given HSA i.g. in MP were significantly greater than those detected in the sera of soluble HSA-treated mice 0, < 0.0002) (Table 3). Indeed, anti-HSA IgGl responses in this latter group were below the limit of ELISA detection. Sera anti-HSA IgGl titres were greatly enhanced in animals given HSA in MP following i.p. antigen boosting, while there was no analogous increase in IgGl responses in the sera of animals primed i.g. with either soluble HSA or buffer alone. Additionally, oral immunization with soluble HSA or HSA-containing TS-PDMS-grafted MP failed to stimulate appreciable HSA-specific sera IgG2a responses following i.g. immunization. Compared to buffer-treated mice, there was also no significant increase in sera IgG2a responses in mice given soluble or microentrapped HSA following a systemic HSA boost. These data support the notion that i.g. immunization with HSA-containing TS-PDMS-grafted MP, but not soluble HSA, selectively primed GALT-derived lymphocytes involved in a systemic humoral recall response following i.p. antigen challenge.

DISCUSSION Mucosal and systemic humoral immune responses can occur independently in response to mucosal and parenteral antigens, respectivelyZ3-“. Nevertheless, antigen administered by either of these routes can modify responsiveness to subsequent immunization by another route, an example being the modulation of mucosal

Priming of GALT with oral microparticles: Table 3 Sera anti-human serum albumin (HSA) reciprocal end-point various forms of HSA, followed by intraperitoneal challenge HSA

titre antibody

TS-PDMS-grafted Soluble HSA NaHCO,

MP

elicited

by intragastric

immunization

with

Post-challenge

Prechallenge lnoculant

responses

P.L. Heritage et al.

IgGl

IgG2a

IgGl

IgG2a

581+194 ND ND

33k19.5 8.6k4.6 ND

26100*7,961 8550 *3,765 3240 k 1,069

499 + 340 578 f 337 195*75

Groups of 10 mice each were immunized i.g. on days 0, 7 and 14 with 50 pg of HSA incorporated into TS-PDMS-grafted MP, HSA in 0.2 M NaHCO, or buffer alone. On day 19, animals were immunized i.p. with 100 pg of HSA solubilized in PBS. Sera obtained on day 15 (prechallenge) or day 33 (post-challenge) were evaluated for the presence of HSA-specific IgGl and IgG2a using an ELISA. Results are expressed as the mean of reciprocal end-point titres representing the greatest serum dilutions giving OD values exceeding twice the buffertreated values f SEM.

antitoxin responses to enterically administered cholera toxin following parenteral cholera toxoid priming. These observations are relevant to immunizing against vaccination will mucosal infections, as parenteral prime” for or boost”.‘2 mucosal immune responses in viva. Despite concern that mucosal immunization might require prior parenteral, antigen exposure to elicit strong circulating humoral immunity, we have demonstrated that i.g. immunization alone stimulates sera antibody responses in the absence of parenteral sensitization4. In our work4, a low antigen dose (lo-50 /fg per mouse) in TS-PDMS-grafted MP, elicited robust sera IgG and IgA responses which, following an oral MP boost, were substantially enhanced. These results suggested that the initial MP immunization protocol provoked humoral recall capability. In the present study, we demonstrated that mucosal MP administration enhanced specific antibody responses in vivo elicited by parenteral boosting. Mice given microentrapped, but not soluble, HSA i.g. demonstrated enhanced HSA-specific sera IgG responses following systemic antigen challenge. Indeed, even i.g. immunization with 50-fold greater doses of soluble HSA failed to stimulate equivalent sera antibody responses following i.p. antigen challenge, thus demonstrating the priming efficacy of i.g. administered TS-PDMS-grafted MP for systemic antigen challenge. This study strongly suggests that the i.g. administration of HSA-containing MP stimulated mucosal immunity via PP. Following i.g. immunization with a low dose of HSA-containing MP, but not soluble HSA, antigen-specific proliferal:ion of PP cells was noted and reached a maximum after a third i.g. immunization. In contrast, antigen-specific lymphocyte proliferation was not observed in gut lamina propria lymphocytes following i.g. immunizati’on with MP (data not shown). These results are consistent with those found by others ‘7~‘8~‘“-2z demonstrating particulate antigen uptake into PP. HSA-specific IgM-secreting SFCs were initially detected in PP cell isolates following a single i.g. MP immunization anld increased following three i.g. immunizations. These results support our observation that three oral MP immunizations are required to elicit maximal sera antibody titres and that oral immunization with soluble HSA fails to generate sera antibody responses (data not shown). We demonstrated that i.g. immunization with HSA-containing TS-PDMS-grafted MP generated systemic humoral immunity in vivo which was augmented by a parenteral antigen boost. These results

suggested PP-stimulated lymphocytes might have migrated to systemic lymphoid compartments following i.g. MP administration. This hypothesis was supported by demonstrating lymphocyte activation in PP following i.g. MP administration and the subsequent appearance of lymphocyte proliferation in MLN and splenic tissue. We observed a shift in specific IgM SFCs from PP and MLN cell isolates to SPL tissue following i.g. MP immunization. The paucity of specific IgG SFCs in PP, MLN or SPL cells shortly after i.g. MP administration suggests gradual B cell maturation in PP, resulting in isotype switching and migration of IgG-committed plasmacytes from the GALT to systemic sites. Peyer’s patches, and possibly MLN, could serve as depots for oral administered HSA-containing MP17,‘“. Although others have demonstrated that orally administered MP might directly stimulate systemic lymphoid tissues following transport through the mesenteric lymphatic?, the time delay between PP and SPL cell activation in our studies suggests that MP or released HSA is not directly stimulating systemically-situated lymphocytes. Rather, orally administered MP might accumulate in the GALT, resulting in sustained stimulation and eventual emigration of IgG-committed HSA-specific lymphocytes. However, it also possible that the observed systemic priming resulted from trafficking of MP-containing antigen-presenting cells (APCs) from the GALT to systemic sites, where specific lymphocytes could then be activated. This notion is supported by data demonstrating both the phagocytosis of particulates by a variety of APCs including B lymphocytesZ7, macrophages’7328.29,dendritic cells3”.3’,and the migration of GALT-derived APCs to peripheral sites”8,32-34. Whereas oral administration of HSA-containing MP primed mice for an enhanced HSA-specific sera IgG (in particular IgGl) response following a parenteral HSA boost, specific circulating IgA levels were not enhanced (data not shown). This is in contrast to our previous study demonstrating enhanced sera IgG and IgA responses following an oral antigen challenge4. These results suggest that MP-stimulated IgA-committed PP lymphocytes are not restimulated following i-p. HSA challenge, a finding consistent with the view that mucosally-stimulated plasmacyte precursors are known to selectively localize to mucosal, but not to systemic sites’. Indeed, after i.g. MP immunization, abundant numbers of HSA-specific SFCs were detected in PP and MLN cell isolates; however, no specific IgA SFCs were detected in SPL

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Priming of GALT with oral microparticles:

P.L. Heritage et al.

preparations up to 28 days following i.g. immunization (data not shown). Our data supports the notion that oral immunization with microentrapped HSA stimulates a subset of Th cells, hallmarked by the appearance of IL-4-secreting lymphocytes (Table 2). The cells are classified into two subsets depending on their cytokine profile. Thl cell clones exclusively produce IL-2, IFN-p, and help to generate and lymphotoxin IgG2a responses, whereas Th2 cells synthesize IL-4, IL-5, IL-6 and IL-10 and provide help in mounting IgA, IgE and IgGl response?s.36. Although Xu-Amano ef a1.37 demonstrated that mature PP T cells are multipotent and can become either Thl or Th2 cells following polyclonal T-cell activation in vitro, they and others have demonstrated also that antigen delivey_to the GALT preferentially stimulates Th2-type cells7 ‘9. Our results confirm and extended previous work”, demonstrating a shift of Th2-type cytokine secretion from GALT (PP) to systemic (SPL) lymphoid compartments with HSA-containing following i.g. immunization TS-PDMS-grafted MP (Table 2). This cytokine microenvironment was likely responsible for driving the predominately IgGl sera response observed after i.g. MP administration, which was boosted following the parenteral antigen boost (Table 3), perhaps via migration of PP-derived T cells. Although the mechanism responsible for the observed MP-induced Th2 cell activity is unknown, recent studies suggest that both and other non-T T-ccl I precursors4” naive IL-4-producing cells4’,“’ could be responsible for initiating differentiation of Th2 cells from multipotent precursors. TS-PDMS-grafted MP are a useful addition to mucosal and systemic vaccine delivery systems. Mucosal priming with these novel polymer-grafted starch MP might improve current systemic vaccine protocols by stimulating elements of the common mucosal immune system in addition to potentially reducing the need for repeated systemic vaccination boosts. Studies are currently under way in our laboratory to investigate the mechanism of MP-induced antigen processing and presentation by gut-associated antigen-presenting cells.

6

7

8

9

10

11

12

13

14

15

16

17

18

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