Vaccine 24 (2006) 5552–5558

Milk IgA responses are augmented by antigen delivery to themucosal addressin cellular adhesion molecule 1

Susan Johnson a,c, Dorothee Bourges a, Odilia Wijburg b,Richard A. Strugnell b, Andrew M. Lew a,∗

a The Walter and Eliza Hall Institute of Medical Research and the Co-operative Research Centre for Vaccine Technology,1G Royal Parade, Parkville 3050, Vic., Australia

b Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology,University of Melbourne, Parkville 3010, Vic., Australia

c Department of Medical Biology, University of Melbourne, Parkville 3010, Vic., Australia

Received 9 September 2005; received in revised form 14 March 2006; accepted 21 April 2006Available online 4 May 2006

Abstract

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The mucosal addressin cellular adhesion molecule 1 (MAdCAM) is expressed on the venules of the gut associated lymphoid tissue (GALT);t is also expressed on the venules of the lobules of the mammary gland. We have previously found that MAdCAM-targeting using a rat anti-

AdCAM monoclonal Ab as both antigen and targeting moiety resulted in an enhanced local IgA gut response. We therefore surmisedhat such targeting may also enhance IgA responses in the mammary gland. We show that our model antigen localizes to the lobules of the

ammary glands as well as the GALT, but not to the draining lymph nodes and that targeting MAdCAM results in secretory IgA responsesn the milk. We provide evidence that this milk IgA Ab is of a secretory nature and is consistent with derivation from gut plasmablasts thatave migrated to the mammary gland. Targeting MAdCAM may be a way for a novel vaccine strategy that affords protection to the mammaryland and the suckling neonate.

2006 Elsevier Ltd. All rights reserved.

eywords: MAdCAM; Milk IgA; Targeting; GALT

. Introduction

Lymphocytes primed by antigen in the gut associated lym-hoid tissue (GALT) up-regulate expression of �4�7 integrin,hich interacts with its receptor mucosal addressin cellu-

ar adhesion molecule 1 (MAdCAM) and causes them toxtravasate to mucosal tissues. MAdCAM is expressed on theurface of high endothelial venules (HEV) in the GALT andat endothelial venules in the lamina propria. Cells primed

n peripheral lymphoid organs express the �4�1 integrin andence do not lodge in mucosal tissues [1]. This differenceetween those primed in the gut expressing �4�7 and thoserimed parenterally expressing �4�1 explains why injectedaccines lack efficacy against mucosal pathogens. MAdCAM

∗ Corresponding author. Tel.: +61 3 9345 2463; fax: +61 3 9347 0852.E-mail address: lew@wehi.edu.au (A.M. Lew).

is also expressed on the endothelial cells of small venulesaround mouse mammary lobules during lactation [2,3]. Thelevels of MAdCAM in the mouse mammary gland increasefrom day 7 of gestation, peaking near parturition and subsid-ing at weaning [2]. Thus MAdCAM is thought to participatein directing lymphocyte traffic into the lamina propria of theintestines and lactating mammary gland [4].

IgA present in colostrum or milk can offer passive pro-tection for the gastrointestinal system of the newborn [5]. Inhumans, the IgA is not absorbed by the neonate, but protectsmucosal surfaces from infection; it can also influence theintestinal flora and the development of the infant’s immunesystem [6,7]. Neonates are born hypogammaglobulinaemic[8] and human babies are reliant on the protection given bymaternal antibodies for the first 12 months after birth [6].

We have previously described a MAdCAM-targeting strat-egy that uses monoclonal antibodies (mAb) of rat IgG2a

264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.04.029

S. Johnson et al. / Vaccine 24 (2006) 5552–5558 5553

isotype against MAdCAM (MECA367; thus it serves bothas targeting moiety and as antigen) or a non-targeted con-trol (GL117) to deliver antigens directly to the mucosal sites[9]. Targeting these antigens to MAdCAM induces a highfaecal IgA response as well as a high serum IgG response.The antibodies are delivered intravenously, but are targeted tothe GALT. This systemic vaccination for mucosal immunitybypasses the formidable hurdles that encounter antigens inthe alimentary lumen. As MAdCAM is also expressed in thevenules around the mammary alveoli [2], we surmised thatthis targeting strategy may also be useful for increasing IgAlevels in the milk of lactating mice.

2. Materials and methods

2.1. Mice

Mice were bred in specific pathogen free conditions in theanimal facilities of Walter and Eliza Hall Institute of MedicalResearch, Parkville, Australia. Female BALB/c and CBA/Hmice strains, 5–8 weeks of age, were used for the experiments.

Both strains behaved similarly in our experiments. Poly-meric immunoglobulin receptor knock-out mice (pIgR−/−)were generated on a C57Bl/6 background [10].

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tocin, Troy Laboratories, Smithfield, NSW, Australia) wasinjected subcutaneously, and then mice were anaesthetizedwith Penthrane. Milk was collected with a pipette onto iceand centrifuged at 6000 × g and 4 ◦C. The whey was col-lected from below the fat with a syringe and used in ELISAsto test for Ab.

2.5. Histology

Tissues (e.g. Peyer’s patches, mesenteric lymph nodes,inguinal lymph nodes draining the lactating mammary gland,lactating mammary gland) were embedded in OCT medium(Sakura, Tokyo, Japan) and frozen immediately on dry ice.Cryostat sections (5 �m) were cut onto polylysine coatedslides (Menzel-Glaser, Germany) then fixed in cold acetonefor 10 mins. Mammary gland sections were cut at −30 ◦C;this lower temperature facilitates sectioning loose tissue. Theslides were stored at −20 ◦C until use. Before immunos-taining, the slides were hydrated in PBS, and blocked with2% skim milk powder and 10% FCS in PBS. Sections werestained with anti-rat Ig-FITC absorbed against mouse Ig(Silenus, MI, USA) at a concentration of 1 in 1000 for anhour in the dark.

2.6. Ab detection by enzyme-linked immunosorbentassay (ELISA)

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.2. Immunizations

Tail vein, quadriceps and flank were used as sites of intra-enous, intramuscular and subcutaneous injections, respec-ively. The antigens used were an anti-MAdCAM mAb

ECA367, anti-MAdCAM mAb MECA 89 or a rat IgG2asotype control GL117 (originally from Dr J. Abrams,NAX), which recognizes bacterial �-galactosidase. Theose used was 50 �g. These mAb were either purchased fromD Biosciences (San Jose, CA, USA) or purified in house

rom hybridoma supernatants.

.3. Preparation of faecal samples

Mucosal antibody (Ab) was isolated from faecal sam-les as a measure of gastrointestinal immune responses.resh faecal samples were collected by separating mice intolean boxes and collecting about three pellets directly intoicrocentrifuge tubes containing 200 �l of 0.1 mg/ml trypsin

nhibitor (Sigma, St. Louis, MO, USA) kept on ice. Sam-les were weighed and 200 �l of trypsin inhibitor was addeder 0.02 g of faeces. Samples were then shaken in a mini-eadbeater (Biospec Products, Bartlesville, OK, USA) for0 s at 2500 rpm and rotated at 4 ◦C for 1–2 h. The supernatantas clarified by centrifugation at 6000 × g 4 ◦C for 20 min.

.4. Collection of milk

Milk samples were collected from lactating mice 7 daysfter parturition. Oxytocin (0.1 ml of 10 IU/ml; Ilium syn-

Rat IgG2a specific Ab responses from serum, faecal andilk samples were determined by ELISA. Microtiter plates

Dynatech, Chantilly, VA, USA) were coated with antigen4 �g/ml GL117 in PBS) for 2 h or overnight at 4 ◦C. Sam-les were added at a concentration of 1 in 10 for faecal andilk extracts and 1 in 25 for serum, serially diluted in blotto

nd incubated for 2 h at room temperature or overnight at◦C. Plates were washed six times between each step withBS/0.05% tween-20. Bound Ab was detected after incuba-

ion with HRP-conjugated antibodies to mouse IgG (donkeynti-mouse, adsorbed against rat Ig; Chemicon, Temecula,A, USA), IgA (goat anti-mouse, Southern Biotechnology,irmingham, AL, USA), diluted in blotto at a concentra-

ion of 1 in 5000. Secretory IgA was detected using aabbit anti-mouse secretory component Ab (courtesy of Drlase Corthesy) and a peroxidase conjugated goat anti-rabbit-

gG secondary Ab (DAKO, Carpinteria, CA, USA). Theubstrate used was tetra methyl-benzidine (T2885, Sigma,t. Louis, MO, USA) in 0.1 M sodium acetate at pH 5.5.he reaction was stopped with 0.5 M sulphuric acid. IgGnd IgA titres were defined as the reciprocal of the high-st dilution to reach an O.D. of 0.1 above background at50 nm.

.7. Total IgA levels by capture ELISA

This was performed as described above except that insteadf GL117, the plates were coated with goat anti-mouse IgA5 �g/ml in PBS).

5554 S. Johnson et al. / Vaccine 24 (2006) 5552–5558

2.8. Statistical analysis

Student t-test was used throughout.

3. Results

3.1. Targeting MAdCAM in pregnant mice leads to anincrease in IgA Ab in milk

To investigate the effect targeting MAdCAM would haveon the Ab responses in milk, we injected the rat IgG2a anti-MAdCAM mAb MECA 367 into pregnant mice (7–10 daysafter timed mating). Ab responses in the milk 7 days post-parturition were measured by ELISA against rat IgG2a andcompared against the responses from non-targeted isotypecontrol. Significant increases in milk IgA responses wereobserved after MAdCAM-targeting when compared to theisotype control (Fig. 1). Serum IgG and IgA, faecal IgA andmilk IgG were also measured (Fig. 2), showing an increasedresponse in all that were immunized with targeted Ab. Theenhancement of mucosal and systemic Ab responses werealso obtained when using MECA 89, an anti-MAdCAM mAbthat recognizes the second extracellular domain of MAdCAM[11] and hence does not compete with binding by �4�7-bearing lymphocytes (data not shown).

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Fig. 2. MAdCAM-targeting in pregnant mice increases both IgG and IgAresponses in milk, faecal and serum samples. Pregnant mice (eight per group)were immunized with either saline, 50 �g of MECA367 (anti-MAdCAM)or 50 �g of GL117 non-targeted isotype control. Rat IgG2a specific Abresponses for milk, faecal and serum IgG and IgA were measured by ELISA.Mean ± S.D. log Ab titres are shown.

by MAdCAM-targeting were similar for all three routes.As expected, no response was elicited without MAdCAM-targeting for all three routes (Fig. 3).

3.3. MAdCAM-targeting in milk has no effect on totalIgA levels

As well as detecting IgA antibodies against rat IgG2a, wewanted to test whether the MAdCAM-targeting has any effecton total IgA levels in the milk. As expected total IgA levelsshowed no change between targeted and non-targeted groups(Fig. 4); therefore, there was no polyclonal activation of IgAsecreting B cells.

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.2. All parenteral routes are equally effective

To determine whether the route of administration has anyffect, we immunized pregnant mice with both the targetingnd non-targeting Ab using subcutaneous, intramuscular andntravenous routes. The enhancement of milk IgA responses

ig. 1. MAdCAM-targeting in pregnant mice induces IgA response. Preg-ant mice (eight per group) were immunized i.v. with either saline, 50 �gf MECA367 (anti-MAdCAM) or 50 �g of GL117 non-targeted isotypeontrol. Rat IgG2a specific Ab responses for milk IgA were measured byLISA.

ig. 3. IgA response in milk is increased regardless of immunization route.regnant mice, five per group, were immunized subcutaneously (s.c.), intra-enously (i.v.) or intramuscularly (i.m.) with 50 �g of either MECA367 (anti-AdCAM) or GL117 (non-targeted isotype control) in saline. Rat IgG2a

pecific Ab responses for milk IgA were measured by ELISA. Mean ± S.D.og Ab titres are shown. *P = 0.02 and **P = 0.015, respectively; t-test.

S. Johnson et al. / Vaccine 24 (2006) 5552–5558 5555

Fig. 4. MAdCAM-targeting does not alter total IgA in milk. Pregnant mice(eight per group) were immunized with either saline, 50 �g of MECA367(anti-MAdCAM) or 50 �g of GL117 non-targeted isotype control. Levels ofnon-specific total IgA were measured by ELISA. Mean ± S.D. log Ab titresare shown.

Fig. 5. The IgA response elicited in MAdCAM-targeting is that of secretoryIgA. Pregnant mice (eight per group) were immunized with either saline,50 �g of MECA367 (anti-MAdCAM) or 50 �g of GL117 non-targeted iso-type control. Levels of secretory IgA were detected by ELISA using asecondary anti-SC Ab. Mean ± S.D. log Ab titres are shown.

3.4. MAdCAM-targeting and secretory IgA

As the majority of mucosal Ab responses are of a secre-tory nature [2,12], we anticipated that the response in themilk should be of secretory nature. A rabbit Ab against mousesecretory component was used to detect secretory IgA. Secre-tory IgA was found in high titres from the milk of those micetargeted with MAdCAM (Fig. 5).

As secretory IgA is formed from the cleavage of the trans-membrane portion of the polymeric immunoglobulin receptor

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ig. 6. IgA levels in PIgR deficient mice. (a) Four pregnant PIgR deficient mice wereesponses for milk, faecal and serum IgA were measured by ELISA and compareatios of milk to serum and faecal to serum IgA for anti-rat Ab and for total IgA le

immunized with MECA367 (anti-MAdCAM). Rat IgG2a specific antibody

d against wild-type mice. Mean ± S.D. log antibody titres are shown. (b)vels in wild-type and PigR−/− mice.

5556 S. Johnson et al. / Vaccine 24 (2006) 5552–5558

Fig. 7. Total milk IgA levels in naıve PIgR deficient mice. Levels of totalmilk IgA were measured by capture ELISA in both PIgR deficient mice andwild-type mice. Mean ± S.D. log Ab titres are shown.

(pIgR) after it has associated with IgA, we wanted to testMAdCAM-targeting in pIgR−/− mice. There was no pub-lished information on IgA levels in the milk of pIgR−/−mice. Milk, faeces and serum were tested. As expected, inthe absence of pIgR, there are extremely high levels of IgAin the serum. As faecal IgA is all secretory IgA, it was notsurprising that there was no IgA Ab detected in the faeces ofpIgR−/− mice (Fig. 6a). In contrast, a fraction of milk IgA,albeit modest, comes from the circulation (cf. 90% is tran-scytosed secretory IgA) [12], the high levels of serum IgAAb in PigR−/− mice led to milk IgA Ab to be comparableto that of wild-type mice (Fig. 6a); this is also reflected inthe levels of total IgA in the milk (Fig. 7). In contrast, thisIgA Ab level is only equivalent to about 1% of serum lev-els (Fig. 6b), whereas in wild-type mice, milk IgA Ab levelswere >20-fold greater than serum levels.

3.5. Antigen localizes to the venules of the of lactatingmammary lobules but not to the draining lymph nodes ofthe mammary gland

To visualize the localization of the targeting Ab, sectionswere taken two days after immunizing lactating mice withtargeting or non-targeting Ab and stained with fluorescent(FITC) tagged anti-rat Ig Ab. As expected, fluorescence wasom

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patches and mesenteric lymph nodes, but not to the draininglymph nodes of the mammary glands (Fig. 8). Moreover, wehave previously found by ELIspot assay that Ab secretingcells were found in the GALT after MAdCAM-targeting [9],but we were unable to detect such cells in the draining lymphnodes of the lactating mammary gland (data not shown). Thisindicated that the source of IgA secreting cells did not origi-nate from the draining lymph nodes.

4. Discussion

We found that MAdCAM-targeting in pregnant miceincreased Ab responses for serum IgG and IgA, gut IgA andmilk IgA when compared with those immunized with theisotype control. The milk IgA Ab was secretory in nature.There is no MAdCAM expression in the lymph nodes drain-ing the mammary gland (lactating or dry) and injected anti-MAdCAM did not localize there. Therefore, the origin ofthis secretory IgA does not seem to be from the lymph nodesdraining the mammary gland. The increase in IgA is not dueto any unintended blocking effects of the MECA367 anti-MAdCAM antibody, as the same effect is observed whenusing the MECA 89, an anti-MAdCAM mAb which cannotcompete against �4�7 lymphocyte trafficking as it binds adifferent domain [11]. The most likely explanation is that�tmtotimtgteho

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bserved in venule-like structures in the lobules of the mam-ary gland (Fig. 8d; [3]).To determine whether IgA Ab-secreting cells may have

een elicited in the draining nodes of the mammary gland,e also performed the antigen localization studies on such

issues. We observed that the antigen localizes to the Peyer’s

4�7 integrin-bearing gut B cells primed by MAdCAM-argeting [9] home to MAdCAM-expressing venules in the

ammary lobules. The homing of plasmablasts/B cells fromhe intestine to the lactating mammary gland has been previ-usly described [13] and there are many reports that suggesthat IgA secreted in milk reflects antigenic exposure in thentestine [13–15]. This link between the immunity of theammary gland and that of the gastrointestinal system is

eleologically sound, as it represents a way of transferringut immunity against the organisms encountered by the damo the offspring which would usually be exposed to the samenvironment (and hence similar organisms) as the dam. Thisoming of gut lymphocytes to the mammary gland occursnly during lactation [16].

An important factor in vaccination strategies is the deliv-ry route; as intravenous delivery is highly impractical foroutine vaccination purposes, both subcutaneous and intra-uscular routes were also used. All three routes (i.m., i.v.,

.c.) were equally efficient in the pregnant mice at eliciting ailk IgA response against IgG2a. The isotype non-targeted

ontrol did not elicit milk IgA responses by any of the threeifferent routes of delivery. The subcutaneous and intra-uscular routes make this strategy applicable to common

accination procedures.In the absence of the polymeric immunoglobulin receptor

pIgR), IgA is unable to be translocated across the epithe-ial cells; thus, the only IgA in peripheral parenchyma iserived from transudation from blood plasma [10]. Hence,s expected, without translocation across the gut wall, fecalgA levels in the PIgR−/− mice were negligible. Conversely,

S. Johnson et al. / Vaccine 24 (2006) 5552–5558 5557

Fig. 8. Antigen localizes to Peyer’s patches and mesenteric lymph nodes but not to the draining lymph node of the mammary gland. Lactating mice wereimmunized with 50 �g of either MECA367 (anti-MAdCAM) or GL117 (non-targeted isotype control). Two days post immunization sections of Peyer’s patches(a), draining lymph node of the mammary gland (b), mesenteric lymph nodes (c) and mammary glands (d) were stained with FITC conjugated anti-rat Ig.

5558 S. Johnson et al. / Vaccine 24 (2006) 5552–5558

the serum IgA levels were extremely high (1000-fold abovewild-type), presumably from the accumulation of IgA thatwould have normally translocated into the gut lumen. How-ever, the milk IgA Ab levels were similar in the wild-type andpIgR−/− mice. Like the Ab levels, total IgA in the milk ofPIgR−/− mice were similar to that of wild-type mice. Thiscontrasted with the absence of IgA Ab in faeces and at firstglance seemed surprising. However, the levels of milk IgAAb in PIgR−/− mice only represents 1% of serum levels.Even though only 10% of milk IgA is normally of serumorigin [12], the high levels of serum IgA in PIgR−/− micemeant that transudation from such high levels of serum IgAwould result in substantial levels of milk IgA in such mice.

In conclusion, targeting MAdCAM enables delivery ofantigen to both the mucosa and the mammary glands,enabling both systemic and mucosal immunity to beincreased. In the early stages of infant life, stimulating gutimmunity to combat enteric infections can reduce mortal-ity rates, and stimulation of local gut immunity can alsoinfluence the homeostasis of the flora colonizing the gut[17,18]. MAdCAM-targeting could be used to vaccinate themother instead of the neonate, to transfer protection to theneonate via milk IgA. This may be particularly useful whereimmunity needs to be immediate or where vaccines elicitpoor responses in the young. The increased levels of milkAb offer promises of protection to newborn infants againsteoamawahta

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[2] van der Feltz MJ, de Groot N, Bayley JP, Lee SH, VerbeetMP, de Boer HA. Lymphocyte homing and Ig secretion inthe murine mammary gland. Scand J Immunol 2001;54(3):292–300.

[3] Nishimura T. Expression of potential lymphocyte trafficking medi-ator molecules in the mammary gland. Vet Res 2003;34(1):3–10.

[4] Berlin C, Berg EL, Briskin MJ, et al. Alpha 4 beta 7 integrinmediates lymphocyte binding to the mucosal vascular addressinMAdCAM-1. Cell 1993;74(1):185.

[5] Weisz-Carrington P, Roux ME, Lamm ME. Plasma cells and epithe-lial immunoglobulins in the mouse mammary gland during preg-nancy and lactation. J Immunol 1977;119(4):1306–7.

[6] Zinkernagel RM. Maternal antibodies, childhood infections,and autoimmune diseases. N Engl J Med 2001;345(18):1331–5.

[7] Hanson LA, Korotkova M, Telemo E. Breast-feeding, infant for-mulas, and the immune system. Ann Allergy Asthma Immunol2003;90(6 Suppl. 3):59–63.

[8] Van de Perre P. Transfer of antibody via mother’s milk. Vaccine2003;21(24):3374–6.

[9] McKenzie BS, Corbett AJ, Johnson S, et al. Bypassing luminal bar-riers, delivery to a gut addressin by parenteral targeting elicits localIgA responses. Int Immunol 2004;16(11):1613–22.

[10] Uren TK, Johansen FE, Wijburg OL, Koentgen F, Brandtzaeg P,Strugnell RA. Role of the polymeric Ig receptor in mucosal B cellhomeostasis. J Immunol 2003;170(5):2531–9.

[11] Streeter PR, Berg EL, Rouse BT, Bargatze RF, Butcher EC. A tissue-specific endothelial cell molecule involved in lymphocyte homing.Nature 1988;331(6151):41–6.

[12] Hanson LA, Korotkova M, Lundin S, et al. The transfer of immu-

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nteric infections as well as contributing to mucosal home-stasis. MAdCAM-targeting as a vaccinating strategy couldlso confer protection against infections of the lactating mam-ary gland. Mastitis is a common problem among women

nd dairy animals [19,20]. Mastitis develops in 5–33% ofomen at some time during lactation. The majority of cases

re caused by S. aureus [21], but for both dairy animals andumans no protective antigen has been identified. MAdCAM-argeting may be useful for improved testing of candidatentigens and for the development of a mastitis vaccine.

cknowledgements

We thank the Australian Government for financial sup-ort through the Co-operative Research Centre for Vaccineechnology and through the National Health and Medicalesearch Council. O.W. is the recipient of a Roper fellowship..J. is the recipient of a Melbourne University scholarship.

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