Bypassing luminal barriers, delivery to a gut addressin by parenteral targeting elicits local IgA responses

Brent S. McKenzie1,5, Alexandra J. Corbett1, Susan Johnson1, Jamie L. Brady1, Jill Pleasance1, David R. Kramer2, Jefferey S. Boyle3, David C. Jackson4, Richard A. Strugnell4 and Andrew M. Lew1

1 The Walter and Eliza Hall Institute of Medical Research and Co-operative Research Centre for Vaccine Technology, 1G Royal Parade, Parkville 3050, Australia
2 Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood 3125, Australia
3 CSL Ltd, 45 Poplar Road, Parkville 3052, Australia
4 Department of Microbiology and Immunology, University of Melbourne, Parkville 3010, Australia
5 Present address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304, USA

Correspondence to: A. M. Lew; E-mail: lew{at}wehi.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of mucosal immunity, particularly to subunit vaccines, has been problematic. The primary hurdle to successful mucosal vaccination is the effective delivery of vaccine antigen to the mucosal associated lymphoid tissue. Physical and chemical barriers restrict antigen access and, moreover, immune responses induced in the mucosa can be biased towards tolerance or non-reactivity. We proposed that these difficulties could be circumvented by targeting antigen to the gastrointestinal associated lymphoid tissue via systemic (parenteral) rather than alimentary routes, using antibodies specific for the mucosal addressin cellular adhesion molecule-1 (MAdCAM). After intravenous or intramuscular injection of such rat antibodies in mice, we found a greatly enhanced (up to 3 logs) anti-rat antibody response. MAdCAM targeting induces a rapid IgA antibody response in the gut and vastly improves the systemic antibody response. Targeting also enhanced T cell proliferation and cytokine responses. Parenteral targeting of mucosal addressins may represent a generic technique for bypassing mucosal barriers and eliminating the need for adjuvants in the induction of proximal and systemic immunity.

Keywords: adhesion molecules, antigen targeting, mucosa, vaccination


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Most infections [including those by Helicobacter pylori, rotavirus, influenza and sexually transmitted human immunodeficiency virus (HIV)] begin at a mucosal site. To stop these infections at this first line of defence, much effort has been devoted to the development of effective mucosal vaccines and adjuvant systems (1, 2). In the gastrointestinal tract, antigen stimulation within the gastrointestinal associated lymphoid tissue (GALT) [such as the Peyer's patches (PP) and mesenteric lymph nodes (MLNs)] induces secretory IgA antibody, CD4+ and CD8+ T cell responses. Reaching this goal of mucosal protection, however, has proven difficult. The gastrointestinal tract imposes a host of formidable obstacles including mucus barriers, degradative gastric acid, alimentary enzymes, bile salts and commensal microflora (3, 4). The amount of antigen that finally reaches the underlying mucosal lymphoid tissue either is ignored or induces tolerogenicity; thus, unwanted responses to the heavy burden of dietary and other benign antigens are effectively blocked. Indeed, this tolerogenic bias forms the foundation for oral and intra-nasal interventions against autoimmune disease (57). Overcoming these barriers has been the principal hurdle in the development of effective vaccines against gastrointestinal pathogens.

Whereas there are many commercial injectable vaccines that work systemically (e.g. measles, mumps, rubella, diphtheria, pertussis, tetanus, Haemophilus influenzae B (hiB), hepatitis B and varicella), there are very few mucosal ones (of the many routine childhood vaccines, there is only one that is mucosally administered, oral polio) (8). There are no killed or subunit mucosal vaccines that are licensed. This is particularly salient because of the perceived litigation risk of live vaccines highlighted recently by unexpected intussusception associated with a live rotavirus vaccine which was consequently withdrawn (9). Moreover, live oral vaccines can be less immunogenic in developing countries, presumably from competition by enteric viruses or helminths (10, 11).

Systemic immunization seldom leads to a local mucosal response. For example, the Salk polio vaccine which is injected does not result in mucosal immunity (12). Direct injection into mucosal lymphoid tissues can induce a mucosal response experimentally (13, 14) but is clinically impractical. Delivery with either biological (15) or synthetic carriers (16) has not been reliably efficacious. Enterobacterial toxins (17) [e.g. cholera toxin (CT)] have been commonly used as experimental mucosal adjuvants; however, as little as 5 µg causes severe diarrhoea in humans (9). With the clinical promise of these approaches unmet, there remains an ongoing need to develop alternative technologies for the delivery of mucosal vaccines.

The mucosal lymphocyte-homing receptor [mucosal addressin cellular adhesion molecule (MAdCAM)] is present on the high endothelial venules (HEVs) of the MLN and PP and in the flat vascular endothelium of the lamina propria (LP) (18, 19). Lymphocytes use MAdCAM to home from the blood to the GALT through {alpha}4ß7 integrins (20). We propose a MAdCAM-targeting strategy that uses the haematogenous (inside) rather than luminal (outside) route to localize antigen to the GALT, thereby bypassing the need for antigen to penetrate through the mucous membranes or contend with the harsh conditions of the alimentary lumen. Rat IgG2a was used as our model antigen; thus, the mAb specific for mouse MAdCAM (e.g. MECA-367) would target to the mucosal addressin and an isotype control would not. Using this simple model (outlined in Fig. 1), we show here that parenteral delivery of MAdCAM-targeted antigen bypasses the luminal barrier to induce potent mucosal and systemic responses.



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Fig. 1. Scheme of systemically targeting the vascular addressin MAdCAM that is specifically found on the venules of the GALT, thereby circumventing mucosal barriers. Rat anti-MAdCAM mAbs (MECA-367 and MECA89) were used for targeting MAdCAM in MLN and PP of the GALT. The control was an irrelevant rat IgG2a isotype (GL117). The read-out used the GL117 as antigen on ELISA or T cell assays.

 

    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immunizations
The mAb immunogens used were a rat IgG2a mAb against mouse MAdCAM (MECA-367, PharMingen, San Diego, CA, USA), a rat IgG2a mAb against mouse follicular dendritic cells (FDCs) (FDC-M2, ABS Biotechnology, Oxford, UK), a rat IgG2a isotype control (GL117 that recognizes bacterial ß-galactosidase), anti-mouse peripheral lymph node addressin (PNAd) (MECA-79, PharMingen) and its isotype control (IR473, Serotec, Raleigh, NC, USA). These mAbs were purchased from the supplier or isolated in-house from hybridoma supernatants and purified on immobilized protein G (Amersham Pharmacia Biotech, Little Chalfont, UK). CBA mice (6- to 8-week old female; 5–10 per group unless otherwise stated) were used for all experiments. Oral immunizations were performed by gavage of 0.3 ml of antigen in 3% wt/vol sodium bicarbonate buffer after light anaesthesia with methoxyflurane (Medical Developments, Springvale, Australia). Subcutaneous immunizations were given in 0.05- to 0.1-ml volumes by injecting into the base of the tail. Intravenous immunizations were performed by injecting into the tail vein in 0.2 ml unless otherwise stated. Intramuscular immunizations were performed by injecting into the quadriceps (0.1 ml into each quadriceps) after light anaesthesia. For co-immunization experiments ovalbumin peptide (OVA) grade V (Sigma Chemical Co., St Louis, MO, USA) was dissolved in PBS and mixed with targeting or control mAbs. DNA vaccines encoding L-selectin human Ig (hIg) and its control CD5L hIg (the CD5 leader replaces the leader and extracellular regions of L-selectin) were prepared as previously described (21) and immunized intramuscularly as described above. Intra-peritoneal injections were given in 0.2 ml. All immunogens contained <0.06 ng of endotoxin per milligram of antigen, as determined by the Limulus amoebocyte lysate assay (Kinetic-QCL, BioWhittaker, Walkersville, MD, USA).

Mucosal antibody isolation
Faecal.
Mucosal antibody isolated from faecal samples was used as a measure of gastrointestinal immune responses (22). Briefly, 1 ml of 0.1 mg ml–1 soybean trypsin inhibitor (Sigma Chemical Co.) in PBS was added per 0.1 g of faeces and then vortexed in a mini-beadbeater (Biospec Products, Bartlesville, OK, USA) for 10 s at 2500 rpm, debris removed by centrifugation 9000 g, at 4°C, for 15 min and supernatant assayed for antibody.

Radio-iodination
In vivo antigen targeting was demonstrated by radio-tracking with 5 µCi of radio-iodinated protein (specific activity of 40 µCi µg–1; total protein including cold protein = 5 µg). Protein was radiolabelled with I125 by the chloramine T method and injected intravenously. Organs were harvested at 1 h and radioactivity [counts per minute (c.p.m.)] for each whole tissue or six PP was determined on a gamma counter.

Immunohistochemistry
Tissue was harvested into OCT (Sakura, Tokyo, Japan) and frozen immediately on dry ice. Five-micrometre sections were cut onto polylysine-coated slides (Menzel-Glaser, Braunschweig, Germany). Sections were fixed in acetone, rehydrated in PBS and blocked with PBS/10% FCS and then stained with anti-rat Ig–FITC adsorbed against mouse Ig (Silenus, Melbourne, Australia).

Immunological assays
ELISA.
Rat IgG2a, rat IgM, hIg and OVA-specific antibody responses from serum, faecal and culture supernatant samples were determined by ELISA. Briefly, microtitre plates (Dynatech, Chantilly, VA, USA) coated with antigen (2 µg ml–1 in PBS) were incubated with serially diluted sera, faecal extract or culture supernatant (diluted in blocking buffer of 5% skim-milk powder in PBS) overnight at 4°C. Bound antibody was detected after 3 h incubation at room temperature with peroxidase-conjugated antibodies to mouse IgG (donkey anti-mouse, adsorbed against rat Ig; Chemicon, Temecula, CA, USA), IgA (goat anti-mouse), IgG1, IgG2a, IgG2b or IgG3 (rat anti-mouse) (Southern Biotechnology, Birmingham, AL, USA) diluted in blocking buffer. The substrate used was tetramethyl-benzidine (Sigma Chemical Co.) in 0.1 M sodium acetate, pH 6, and reactions were stopped with 0.5 M sulphuric acid. IgG and IgA titres were defined as the reciprocal of the highest dilution to reach an OD450nm of 0.2 and 0.1 above background, respectively. Cytokine levels in the culture supernatant were evaluated by sandwich ELISA. Recombinant cytokines as standards, coating antibody and biotinylated antibody were obtained from PharMingen.

ELISPOT.
To determine the number of cells secreting antibody, ELISPOT assays were performed. Briefly, 96-well sterile multi-screen filtration plates (Millipore S.A., Yvelines Cedex, France) coated with rat IgG2a (GL117, 20 µg ml–1 in PBS) were incubated for 16–36 h at 37°C, 10% CO2, with dilutions of single-cell lymphocyte preparations isolated from bone marrow, MLN, inguinal lymph node (ILN), PP, spleen or LP. LP lymphocytes were isolated as previously described (23). Bound antibody was detected after incubation with peroxidase-conjugated antibodies to mouse IgA or IgG (Southern Biotechnology) diluted in blocking buffer. Numbers of spots representing individual antigen-specific antibody-secreting cells (ASCs) were counted under a stereomicroscope after development with 3-amino-4-ethyl carbazole (AEC) substrate (Dako Co., Carpinteria, CA, USA).

Gastrointestinal explant culture.
Gastrointestinal explant cultures were performed using described methods (23, 24). Briefly, to obtain intestinal segments, PP were excised and the remaining small intestines were stripped of epithelium with 5 mM EDTA, washed and cut into 3-mm2 pieces. Twenty halved PP pieces or 20 intestinal segments were cultured on gelfoam (Amersham Pharmacia Biotech) in 2.5 ml of RPMI with 10% FCS at 37°C, 5% CO2, for 6 days and culture supernatant was used for analysis.

Proliferation and cytokine assays.
T cells were purified on nylon wool (25) and 2 x 105 T cells were cultured with 2 x 105 irradiated splenocytes in a standard 5-day [3H]thymidine-uptake protocol. T cells were determine by FACS to be >90% pure. The mean stimulation index was calculated as the c.p.m. of cells with antigen divided by the c.p.m. of cells without antigen. Supernatant was removed at day 5 for cytokine assays described above.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MAdCAM targeting avoids the oral route to successfully deliver antigens to gut lymphoid tissues in vivo
We employed a radio-tracking technique to examine the localization of antigen within both the systemic and gut mucosal lymphoid compartments after systemic delivery of non-targeted and MAdCAM-targeted antigen. We found that after intravenous delivery of radiolabeled mAbs, radioactivity could be detected in all organs harvested including spleen and peripheral and mucosal lymph nodes (Fig. 2a). However, intravenous delivery of radiolabelled anti-MAdCAM mAb resulted in its preferential localization (over that of the non-targeted isotype control) in the GALT such as MLN and PP, but not in peripheral lymphoid tissues such as ILN (Fig. 2a) or spleen (data not shown). This is consistent with the predominant expression of MAdCAM in mucosal tissues (18, 19). Using immunofluorescent analysis to assess cellular localization, no obvious deposits of the non-targeted mAb could be detected in the GALT (Fig. 2b). In contrast, significant localization of anti-MAdCAM mAb could be easily seen in both the PP and MLN as early as 1 h after immunization (Fig. 2b), consistent with our radiolabeling approach (Fig. 2a). Localization of targeted antigen was restricted to the endothelial venules with no significant deposits of antigen found elsewhere in the tissue (Fig. 2b). Remarkably, in vivo staining of endothelial cells by anti-MAdCAM mAb persisted through 8 days after a single 100-µg dose (Fig. 2b) and remains detectable until 14 days. The reason why this persistence is so remarkable remains unknown. Even though the spleen (sinus lining; marginal zone) does express some MAdCAM, we did not detect any localization of injected anti-MAdCAM antibody. We presume this was due to the relative abundance of MAdCAM in mucosal tissues.



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Fig. 2. Localization. (a) Radio-iodination of proteins revealed preferential localization of MAdCAM-targeted antigen to the GALT in vivo. Binding of anti-MAdCAM mAb MECA-367 was enhanced in MLN and PP compared with the isotype control (*P = 0.013 and **P = 0.002, respectively; t-test). No enhancement was found in peripheral lymphoid tissues (PLT) such as the ILN. Means ± SD c.p.m. are shown. (b) Immunofluorescent analysis revealed antigen localization to the HEVs of the GALT in mice immunized with anti-MAdCAM but not with the isotype control. Staining of HEVs persisted longer than 8 days after a single immunization.

 
Mucosal or parenteral immunization with soluble non-targeted antigen fails to induce mucosal antibody responses
The delivery of purified soluble antigens via mucosal or parenteral routes typically fails to stimulate mucosal antibody responses. It was important to confirm this with our model mAb antigens, as some Igs have immunostimulatory properties. To investigate this, we measured antibody responses to oral and subcutaneous immunization with control isotype rat IgG2a. Antibody responses were measured from serum and faecal samples to indicate systemic and gut mucosal responses, respectively. As we predicted, oral delivery of the antigen in the absence of adjuvant failed to induce a mucosal or systemic antibody response (Fig. 3a). This non-responsiveness could be entirely overcome by addition of CT, a potent mucosal adjuvant. In contrast to this, subcutaneous immunization even with Freund's adjuvant failed to induce a gut IgA response, even though a strong serum IgG response was induced (Fig. 3a). This is typical of parenterally delivered antigens and illustrates the reliance on mucosal adjuvants such as CT for the induction of mucosal responses.



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Fig. 3. Targeting MAdCAM via the systemic route stimulates both potent mucosal and systemic antibody responses. (a) Mice were immunized with 100 µg of rat IgG2a orally with or without 10 µg of CT, or subcutaneously with or without Freund's incomplete adjuvant (FIA). Rat IgG2a-specific antibody responses for fecal IgA, serum IgA and serum IgG were measured by ELISA at 2 weeks. (b–d) Mice were immunized intravenously with 100 µg of either anti-MAdCAM mAb MECA-367 or isotype control mAb GL117 in saline. Rat IgG2a-specific antibody responses for fecal IgA (b), serum IgA (c) and serum IgG (d) were measured by ELISA. Representative data from three experiments (means ± SD of the log titre) are shown.

 
MAdCAM targeting bypasses oral delivery and overcomes the need for adjuvant to stimulate potent gut as well as systemic antibody responses
To investigate whether our parenteral approach to mucosal vaccination (Fig. 1) could bypass the oral route to stimulate gut responses, we immunized mice with either a rat IgG2a mAb specific for MAdCAM (MECA-367) or a non-targeted isotype control (GL117). Remarkably, MAdCAM targeting elicited a strong mucosal response and augmented systemic responses by 2–3 logs (Fig. 3b–d). Mice immunized with non-targeted isotype control mAbs did not develop a faecal IgA response (Fig. 3b), consistent with our previous observations with this antigen. In contrast to this, MAdCAM targeting induced a potent faecal IgA response that peaked at 2 weeks and remained detectable at 8 weeks (Fig. 3b). MAdCAM-targeted immunization did not significantly alter the total level of IgA isolated from the gut, and there was no effect on cell distribution in the GALT (data not shown). Mucosal IgA responses induced by MAdCAM targeting could also be demonstrated in a number of other mouse strains including C57Bl/6 and DBA/1 (data not shown) and were comparable to (or slightly better than) non-targeted antigen delivered orally in CT (Fig. 3a).

In the systemic compartment, antibody responses were also enhanced with MAdCAM targeting. Following similar kinetics to the faecal antibody response, MAdCAM targeting induced a serum IgA response whereas non-targeted isotype control antibody immunization did not (Fig. 3c). The serum IgG response induced by MAdCAM targeting was enhanced 100- to 1000-fold above that without targeting (Fig. 3d).

B cell responses are induced in local effector sites of the GALT
In humans, gut IgA is made locally, but in mice it can be translocated from the blood (26). Although this mechanism only contributes around 0.05% of total fecal IgA (27), we still wanted to determine whether IgA in the fecal samples was of gut origin. A substantial increase in antigen-specific IgA ASC was found in MLN, PP and LP lymphocyte preparations (Fig. 4a). IgA ASC could be detected in the PP and LP as early as 5 days after primary immunization (Fig. 4a), indicating that B cells were stimulated in these sites. The number of specific IgA ASC had increased at day 11 in all three key sites of the GALT (Fig. 4a). Antigen-specific IgA ASCs were not detected in the spleen or bone marrow at 5 or 11 days after immunization (Fig. 4a), indicating that IgA found in the serum was of GALT origin. For further confirmation that IgA was produced locally in the gut, gastrointestinal explant cultures were prepared. IgA antibody could be detected in culture supernatants of PP and intestinal segments from MAdCAM-targeted, but not non-targeted, immunizations (Fig. 4b, inset). Thus, MAdCAM targeting elicits local mucosal IgA responses in the GALT.



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Fig. 4. Cells of GALT origin govern mucosal and systemic antibody responses to MAdCAM-targeted antigen. Mice were immunized intravenously with 100 µg of either anti-MAdCAM antibody MECA-367 or the isotype control GL117. (a) After 5 and 11 days, bone marrow (BM), MLN, PP and LP lymphocytes and spleen cells were harvested and assayed for rat IgG2a-specific IgA ASCs by ELISPOT; means ± SD (spots per 106 cells) are shown. (b) PP and intestinal segments (IS) were taken at 10 days and cultured in vitro for 6 days. Antigen-specific IgA in the culture supernatant was measured by ELISA. (c) After 5 and 11 days, MLN, ILN and PP and spleen cells were harvested and assayed for rat IgG2a-specific IgG ASCs by ELISPOT; means ± SD (spots per 106 cells) are shown. (d) Serum was taken at 2 weeks and IgG subclass response measured by ELISA. Means ± SD are shown.

 
ELISPOT assays were also used to dissect the origin of potent systemic IgG responses. We were unable to detect significant increases in bone marrow, spleen, MLN, ILN or PP IgG ASC 5 days after immunization (Fig. 4c). However, the MLN was the first tissue to show substantial numbers of IgG ASC. Eleven days after immunization, IgG responses were found in MLN but not in the mucosal PP or the peripheral lymphoid tissue of the bone marrow, spleen or ILN (Fig. 4c). These data reflect the major contribution of the MLN to systemic response to MAdCAM-targeted antigen and highlight a potential function of the MLN as the interface between the mucosal and peripheral lymphoid compartments. Enhanced systemic IgG responses induced by targeting were predominantly of the IgG1 isotype, but with significant IgG2a (Fig. 4d, inset). Unfortunately, IgG responses induced by non-targeted immunizations fell below the limit of detection with the isotype-specific reagents. In addition to this, we also found that once mucosal priming occurred after MAdCAM targeting, IgA and IgG responses could be boosted by a secondary intra-peritoneal injection of either non-targeted or targeted antigen; 2 weeks after a 50-µg intra-peritoneal boost, fecal IgA antibody was elevated 3.1- and 4.0-fold, respectively. Thus, once gut responses were primed by targeting, they did not require re-targeting to boost responses in either systemic or mucosal compartments.

MAdCAM targeting is required
To determine whether MAdCAM targeting may lead to bystander effects, we performed co-immunization experiments. We co-immunized mice with another model antigen OVA mixed with either the MAdCAM-targeted (MECA-367) or non-targeted (GL117) rat IgG2a. We chose to deliver 500 µg of OVA as this induces a sub-optimal serum IgG response in CBA mice but is 10-fold higher than that required to prime OVA-specific T cells in mucosal tissues (data not shown). After co-immunization we found that equivalent levels of anti-OVA serum IgG were induced and no anti-OVA IgA responses were elicited in mucosal or systemic compartments (Fig. 5a). Furthermore, co-immunization with OVA had no effect on antibody responses against the rat IgG2a (data not shown). These data support the requirement for direct targeting of antigen to the GALT in the enhancement of mucosal and systemic responses. To explore this further, we analysed the antibody response to antigens targeted to peripheral lymphoid tissues. We chose to explore this by targeting the peripheral counterpart of MAdCAM (PNAd) through both protein (rat anti-PNAd) and DNA (L-selectin fused to human Ig) immunizations. Although targeting PNAd with either approach enhanced the systemic IgG response (over that of the non-targeted controls), they failed to enhance mucosal or systemic IgA (Fig. 5b and c). Because PNAd targeting was not as potent as anti-MAdCAM targeting, it is possible that a stronger anti-PNAd response may have led to a gut IgA response. If this were so, we think this unlikely to be due to potency alone, as the subcutaneous immunization with Freund's adjuvant leads to a very strong serum IgG response but no IgA response (see Fig. 3a). We argue that there is a requirement for the localization of antigen to the GALT for the induction of mucosal responses. Targeting FDCs with the rat IgG2a mAb, FDC-M2, did not elicit an IgA response in serum or gut (Fig. 6d).



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Fig. 5. Antibody responses induced by MAdCAM targeting require mucosal targeting. (a) Mice were immunized intravenously with 100 µg of either anti-MAdCAM mAb MECA-367 or isotype control mAb GL117 mixed with 500 µg of OVA in 0.3 ml of saline. ELISAs were performed 2 weeks after immunization. (b) Mice were immunized intravenously with 100 µg of either anti-PNAd mAb MECA-79 or isotype control in 0.2 ml of saline. ELISAs were performed 2 weeks after immunization. (c) Mice were immunized intramuscularly at 0 and 6 weeks with 200 µg of a DNA vaccine encoding either L-selectin hIg or CD5 leader (CD5L) hIg control in 0.1 ml of saline (0.05 ml into each quadriceps). ELISAs were performed 8 weeks after immunization. (d) Targeting FDC does not mirror augmented responses induced by targeting MAdCAM. Mice (five per group) were immunized intravenously with 40 µg of either anti-FDC mAb (FDC-M2; solid triangles), anti-MAdCAM (MECA-367; solid squares) or isotype control (GL117; open circles) in saline. All groups were boosted at 8 weeks by intra-peritoneal injection of 100 µg isotype control GL117 in saline. Rat IgG2a-specific antibody responses for fecal IgA, serum IgA and serum IgG were measured by ELISA at 2, 4, 8 and 10 weeks. Means ± SD log antibody titres are shown.

 


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Fig. 6. MAdCAM targeting by all three parenteral routes result in a fecal IgA response. (a) Mice were immunized intramuscularly with 100 µg of either MECA-367 or isotype control GL117 in 0.2 ml of saline (0.1 ml into each quadriceps). Antigen-specific antibody responses for fecal IgA, serum IgA and serum IgG were measured by ELISA at 2 weeks. (b) Mice were immunized with 50 µg of MECA-367 subcutaneously, intramuscularly or intravenously. Fecal IgA ELISA is shown. Means ± SD are shown.

 
Enhanced responses after intramuscular or subcutaneous delivery of MAdCAM-targeted antigen
Parenteral vaccines are usually given via subcutaneous or intramuscular routes. To investigate whether such routes could mirror the responses induced intravenously, we immunized mice intramuscularly with either anti-MAdCAM mAb (MECA-367) or non-targeted control (GL117). We found that intramuscular MAdCAM targeting led to mucosal and systemic IgA responses that were often less efficient but followed the same pattern as responses induced by intravenous route (Fig. 6a). Serum IgG responses were still consistently 100- to 1000-fold higher than the non-targeted control (Fig. 6a). We also compared subcutaneous, intramuscular and intravenous routes and found that all three routes could elicit a fecal IgA response (Fig. 6b).

MAdCAM targeting enhances T cell proliferative and cytokine responses
To assess the T cell response to MAdCAM-targeted antigen, we measured T cell proliferation and cytokine responses after immunization. We found that like the antibody responses, T cell proliferative and cytokine responses were enhanced by MAdCAM targeting (Fig. 7a and b). Splenic T cells purified from mice immunized with non-targeted antigen failed to proliferate in response to antigen (Fig. 7a). In contrast, splenic T cells from mice immunized with MAdCAM-targeted antigen showed a significant proliferative response (Fig. 7a; P < 0.01 at 10 and 100 µg ml–1 antigen concentration). Similarly, MLN T cell proliferation could only be detected in mice that received MAdCAM-targeted immunization (Fig. 7a; P < 0.05 at 100 µg ml–1 antigen concentration). Enhanced secretion of cytokines could also be detected in mice immunized with MAdCAM-targeted antigen (Fig. 7b). Mice immunized with targeted antigen showed elevated production of IFN-{gamma}, IL-2, IL-5 and IL-6, over that of the non-targeted control immunized mice in the systemic response to antigen (Fig. 7b). Augmented responses were also observed in the MLNs that drain the gut. MAdCAM targeting enhances the secretion of IFN-{gamma} and IL-2 by MLN T cells (Fig. 7b). IL-4 responses could not be detected (data not shown).



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Fig. 7. MAdCAM targeting enhances T cell cytokine and proliferative responses. Mice (five per group) were immunized intravenously and boosted intra-peritoneally on day 18 with 100 µg of GL117 in complete Freund's adjuvant. (a) After 10 days, spleens and MLN cells were harvested. Splenocytes and MLN T cells were purified on nylon wool and 2 x 105 T cells were cultured with 2 x 105 irradiated splenocytes in a standard 5-day [3H]thymidine-uptake protocol. The mean stimulation index was calculated as the c.p.m. of cells with antigen divided by the c.p.m. of cells alone. (b) Cytokine levels in the supernatant (from 100 µg antigen recall) were evaluated by sandwich ELISA.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A key step for many pathogens is the invasion or colonization of mucous membranes. Induction of robust immune responses at these sites provides the best chance of escaping infection and preventing its spread. However, induction of protective mucosal responses (particularly to subunit antigens) has proven difficult. In the gut, effective delivery of vaccine antigens to underlying lymphoid tissues has long been recognized as the primary hurdle to the induction of protective responses (4, 28). The most formidable components of this hurdle are the physicochemical barriers. MAdCAM is an ideal target to bypass these barriers as it signposts gut lymphoid tissues in an abundant and relatively specific manner. Fortunately, rat mAbs to mouse MAdCAM are also available. As rat IgG is immunogenic in mice, these mAbs serve as both targeting moieties and model antigens. We found that after systemic delivery of anti-MAdCAM mAbs they preferentially targeted the GALT. Radioactive targeting studies showed an ~2-fold increase in antigen localization in the GALT. This differential is probably a gross underestimate, as we believe that the bulk of the radioactivity found in the non-targeted control was due to blood contamination. This view is supported by the results of the immunofluorescence studies which demonstrated that a non-targeted antigen was undetectable in the GALT. Such studies also showed antigen persistence in the HEV for at least 8 days. Perhaps both preferential localization and antigen persistence (the HEV of the GALT acting as a depot) underpin the enhancements in local gut responses. This model also demonstrates that uptake and delivery of antigens from the gastrointestinal lumen are not required for the induction of local responses. This may provide a useful model for addressing the contribution of M cells and sub-epithelial antigen-presenting cells (APC) in the responses to mucosally delivered antigen. More importantly, we also reveal here that the tolerogenic bias of immune responses in the mucosal associated lymphoid tissue can be overcome without the use of adjuvants by simply controlling the localization of antigen. Moreover, once mucosal responses were induced by targeting, they could be boosted by delivery of non-targeted antigen. We believe that these findings will have significant implications for the design of future vaccine formulations and prime boost strategies.

Targeting also significantly augmented the systemic IgA and IgG responses (2 logs) higher than the non-targeted control. Although MAdCAM is predominantly expressed in mucosal lymphoid tissue, there is some weak expression in peripheral lymphoid sites such as sinus-lining cells and active germinal centres of the spleen (29, 30). Despite this, we found that both IgA and IgG responses were almost exclusively of GALT origin with the MLN as the predominant source of antigen-specific IgG ASC. Consistent with this we found that splenectomized mice responded as well as normal mice to targeted immunizations (data not shown), ruling out any major role for the spleen. We therefore believe that enhanced systemic IgA and IgG responses are the direct result of targeting gut mucosal lymphoid tissues. This is not unusual as local responses induced by mucosal antigen delivery are very often associated with potent systemic responses where the MLNs play a crucial role (3134).

As anti-MAdCAM mAb can interfere with normal lymphocyte homing (19), it was important to dissect the influence of potential bystander effects from that of antigen targeting. Our studies revealed that targeting was indeed required as neither co-immunization of antigen with anti-MAdCAM mAb nor targeting the peripheral node addressins induced a mucosal antibody response. The data concerning the peripheral node addressins also reinforced the tenet that systemic adjuvants that heighten serum IgG responses often do not induce mucosal IgA. Moreover, it highlights that GALT targeting is required for induction of mucosal immunity to parenterally delivered antigen.

Rapid induction of proximal responses is important, particularly when swift protection is required during natural or unnatural (e.g. from biological warfare) outbreaks. The speed of the mucosal response induced by targeting (seen as early as 5 days after a single immunization) combined with the potency of the systemic response demonstrates the potential of this strategy to provide rapid protection in situations where susceptibility is high. Interestingly, mucosal B cell responses were detected at the same time antigen was still present on the mucosal endothelium. It is therefore conceivable that antigen-specific B cells could bind to antigen-labeled endothelium, promoting mucosal lymphocyte homing by enhancing lymphocyte endothelial cell interactions. It is possible that such a mechanism could contribute to enhanced responses induced by MAdCAM targeting.

Although we know that MAdCAM targeting is required, exactly how it improves responses is unclear, but it is likely to be dependent on the traffic of antigen from MAdCAM+ endothelium to mucosal APC. This may occur through two different but not necessarily exclusive mechanisms. The first of these is passive release: it is possible that antigen present on endothelial cells is being released into the surrounding lymphoid tissue. The small trickle of antigen released from endothelial venules into the lymphoid parenchyma may not be detectable in our antigen localization studies. It is interesting that dendritic cells can interact with T cells in the paracortical sites immediately adjacent to the HEVs (35) and hence MAdCAM. Thus, dendritic cells in this location are uniquely positioned to pick up and present MAdCAM-targeted antigen to the waves of incoming lymphocytes and dendritic cells from the blood; 85–90% of lymphocytes entering a lymph node are derived from blood (36). The alternative mechanism of antigen transfer may involve active uptake of antigen from the surface of endothelial cells by incoming cells, as they undergo diapedesis through the venules. For example, blood-derived dendritic cells may cross-present antigens from endothelial cells during homing. It is also possible that antigen transfer is not required and MAdCAM+ cells themselves enhance responses by directly priming or activating lymphocyte subsets. FDCs in the gut also express MAdCAM (37) and it is possible that targeting these cells enhances mucosal antibody responses. Although the exact contribution of targeting MAdCAM+ FDC remains to be elucidated, pan FDC targeting with an anti-FDC mAb does not enhance gut mucosal responses (Fig. 5d). There is, however, growing evidence that vascular endothelial cells are capable of picking up and presenting exogenous antigen (3840). It is therefore possible that vaccine strategies that enhance responses by targeting such cells (including MAdCAM targeting) may do so by enhancing lymphocyte endothelial interactions. The MAdCAM-targeting approach may provide a unique model to further dissect the role of endothelial cells in antigen presentation.

Vaccination strategies that induce potent B cell as well as T cell responses provide the best defence against an invading pathogen (41). Induction of robust T cell responses at mucosal and systemic compartments is considered a crucial ‘checkpoint’ in the development of many vaccines, especially against dangerous pathogens such as HIV. To assess the T cell response in our targeting approach, we measured T cell proliferation and cytokine responses after immunization. We found that both systemic and mucosal T cell proliferative and cytokine responses were enhanced by antigen targeting to MAdCAM. Levels of cytokines important in the induction and maintenance of T cell effector function (IFN-{gamma} and IL-2) and those important in B cell responses (IL-5 and IL-6) were elevated in MAdCAM-targeted mice. However, these responses could only be detected after boosting. Thus it remains moot whether the enhanced T cell proliferation and cytokine responses detected represent direct T cell activation at these sites or the result of homing of primed T cells. We aim to address this question in further experiments using anti-MAdCAM mAbs to target antigens other than rat Ig. This approach will also aid to address the efficacy of MAdCAM targeting in models of mucosal infection.

In summary, we have shown here that targeting mucosal addressins via parenteral routes represents a generic means of overcoming the mucosal barrier and eliminating the need for adjuvants to enhance mucosal and systemic antibody as well as T cell responses. MAdCAM targeting may provide the ideal platform for delivering antigens of gastrointestinal pathogens, such as rotavirus, H. pylori, rectally transmitted HIV, giardia, intestinal helminths and enterobacteria like salmonella. We have established the proof of principle that targeting addressins specific for particular lymphoid compartments can elicit a regional immune response and such strategies may also be applied to target other lymphoid sites draining specific tissues, once a specific addressin has been identified.


    Acknowledgements
 
We thank Prof. Emeritus G. J. V. Nossal (Chairman of the Committee Overseeing WHO Vaccines & Biologicals Programme) for his advice and comments. This work was financially supported by the Ramaciotti Foundation, Appel Estate Fund, Juvenile Diabetes Research Foundation, National Health and Medical Research Council of Australia and the Australian Government via its Co-operative Research Centre Program. B.S.M. was a recipient of an Australian Postgraduate Award.


    Abbreviations
 
AEC   3-amino-4-ethyl carbazole
APC   antigen-presenting cell
ASC   antibody-secreting cell
c.p.m.   counts per minute
CT   cholera toxin
FDC   follicular dendritic cell
GALT   gastrointestinal associated lymphoid tissue
HEV   high endothelial venule
hIg   human Ig
HIV   human immunodeficiency virus
ILN   inguinal lymph node
LP   lamina propria
MAdCAM   mucosal addressin cellular adhesion molecule
MLN   mesenteric lymph nodes
OVA   ovalbumin peptide
PNAd   peripheral lymph node addressin
PP   Peyer's patches

    Notes
 
Transmitting editor: A. Cooke

Received 15 July 2004, accepted 26 August 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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