INTRODUCTION

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Vaccination, conventionally by the parenteral route, has proven to be the most effective means of protecting a population and individuals from infectious disease. More recently, focus on the dangers involved with needle injection, including multiple use, hepatitis, and human immunodeficiency virus infection, and the expense and fear of needles, particularly in Third World countries, have dictated the need for alternative vaccination routes, such as the practical, noninvasive oral route.

Oral delivery requires that the vaccine preparation survive stomach acid and digestive enzymes to arrive intact at the desired site on the digestive tract and to overcome the phenomenon of oral tolerance. Successful delivery is frequently dependent on coadministration of an adjuvant for stimulation of an immune response. Traditionally, orally delivered or mucosal adjuvants have included bacterial ligands and toxins, such as Escherichia coli labile toxin (LT), cholera toxin (CT), and CT B subunit (CT-B) (22). These adjuvants have the ability to bind to intestinal epithelial cells and abrogate oral tolerance to coadministered or covalently coupled antigens (14). However, due to their toxicity, neither CT nor LT is suitable for human use. Alternative mutant toxin molecules (22), including recombinant forms (29) and fusion proteins (1, 30), have been derived, but are often not as effective mucosally when lacking the toxic A1 subunit, with maximal mucosal immunoglobulin A (IgA) responses achieved in the presence of whole CT (13, 22).

Additionally, a number of inert delivery systems, including gelatin capsules (21), microspheres or microparticles (15, 18), and bioadhesive preparations (25), improve vaccine efficacy by allowing intact delivery of the vaccine to the gut mucosa, but are generally without adjuvant activity. Cytokines (2, 8), recombinant bacterial and viral vectors (11, 12), oligodeoxynucleotides (17, 20), immune response-stimulatory complexes (ISCOMS) and lipid derivatives (3, 4, 26), alkyl-polyacrylate esters (16), and other candidate adjuvants and delivery mechanisms (35), including transgenic vegetables (28), have been assessed for mucosal delivery in animal models, with some advancing to human trials. However, the only adjuvants currently approved for human use are aluminum salts and MF59 (24), and the only vaccines routinely used for oral delivery are the live attenuated polio vaccine, the live attenuated Salmonella enterica serovar Typhi vaccine, and the tetravalent rotavirus vaccine, which is currently in doubt (34). Clearly there is much ongoing development of mucosal vaccination strategies. However, the toxicity of existing mucosal adjuvants and the limited range approved for human use, along with other issues (24), including effective dose requirements, stability, and economic measures, warrants additional investigation of putative adjuvants for human administration.

We have previously described an isolate of Giardia duodenalis (BRIS/95/HEPU/2041), referred to as 2041, established from a bird which died of an overwhelming infection with the parasite (31). Isolate 2041 chronically infects mice and produces higher peak parasite loads than the human Giardia isolate BRIS/83/HEPU/106 (referred to as 106) (31, 32). Mice infected with 2041 suffer weight gain impairment, with the most severe weight deficit occurring at the time of maximum parasite load (32). Total serum IgA levels of these mice are threefold higher than those from mice infected with 106 trophozoites, but their specific anti-Giardia serum IgA and IgM levels are significantly decreased (36). Weight loss is often associated with production of toxins by bacteria residing in or in transit through the gut. In general these toxins act by altering electrolyte transport across the intestinal mucosa, resulting in water loss and thus weight loss in animals (23). The toxin gene homologue overexpressed in parasites under drug pressure (10), our Giardia 2041 studies (31, 32, 36), and the body of literature equating some toxins with adjuvants (27, 13) have led to the current assessment of Giardia isolate 2041 adjuvanticity. The toxin gene homologue identified in a G. duodenalis laboratory isolate encodes a protein which has 57% homology with the gene encoding the precursor of the sarafotoxins, a group of snake toxins from the burrowing adder known to cause symptoms similar to those in humans acutely infected with Giardia (10). While identification of this gene in the 2041 isolate is still under investigation, these data demonstrate that Giardia isolates may be capable of producing toxins as part of their infective process, which in turn might display adjuvant activity.

This paper describes initial data on oral administration of antigen with various preparations of Giardia extracts and demonstrates antigen-specific mucosal and systemic adjuvant activity provided by the cytosolic component of Giardia isolate 2041 trophozoites.

	MATERIALS AND METHODS

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Mice. Inbred C57BL/6J, hybrid C57BL/6J × CBA, and outbred Quackenbush 5- to 8-week-old female mice and pregnant Quackenbush female mice were obtained from the Animal Resources Centre (Perth, Australia) and housed under PC2 conditions within Queensland Institute of Medical Research. All protocols were approved by the Bancroft Centre Research Ethics committee, according to National Health and Medical Research Council guidelines.

Parasite culture. Trophozoites of G. duodenalis isolates 2041 (31) and 106 (6) were cultured as previously described (7). Isolate 106 has arbitrarily been designated the standard laboratory isolate.

Parasitic infection. Quackenbush 3-day-old neonates were each infected via intragastric tubing (7) with 5 × 10⁵ trophozoites from either Giardia 2041 or 106 in 50 µl of sterile phosphate-buffered saline (PBS) or with sterile PBS alone, and small intestines were collected at 2 and 3 weeks postinfection. The small intestine was excised from the duodenum to the cecum, and portions of approximately 10 mm in length were removed from the midpoint of this section, frozen in OCT (Tissuetech) embedding agent on dry ice, and then stored at 70°C.

Immunohistochemistry. Frozen samples were sectioned in a microtome at 20°C, mounted on treated (1% 3-aminopropyltriethoxy-saline [Sigma] in acetone) glass microscope slides, then fixed in acetone at 20°C for 10 min, and stored desiccated at 20°C. Fixed sections were brought to room temperature (RT) and rehydrated in PBS for 5 min. Endogenous peroxidases were blocked with 0.8% hydrogen peroxide in methanol solution for 3 to 5 min, and the sections were washed in PBS and then blocked with 0.5% bovine serum albumin (BSA) in PBS for 1 h at RT. The sections were then incubated with either goat anti-mouse IgA-horseradish peroxidase (HRP), IgG-HRP, IgM-HRP, or rat anti-mouse IgE-HRP (Southern Biotechnology Associates Inc.), according to the manufacturer's instructions. The detecting antibody was removed with three washes in PBS, the color was developed using 3,3'diaminobenzidine (Sigma Fast DAB) for 3 to 5 min, and the reaction was stopped with PBS washes. The sections were counterstained with alcoholic eosin for 45 s, dehydrated with ethanol, cleared with three washes of xylene, and mounted in dpx (ProSciTech). Antibody-specific staining was identified by light microscopy.

Antigens and adjuvants. Lyophilized keyhole limpet hemocyanin (KLH) (Sigma) was resuspended in either sodium bicarbonate (pH 9.0) or sterile PBS at the time of inoculation. Inactivated influenza virus (Flu) strain A/Texas/36/91 (3.25 mg ml¹) was supplied by Lorena Brown (Department of Microbiology and Immunology, Melbourne University, Melbourne, Australia) and stored at 4°C. CT (Sigma) from Vibrio cholerae was reconstituted to 2 mg ml¹ using sterile Milli-Q water and stored at 4°C.

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View larger version (62K): [in this window] [in a new window]   FIG. 1.   Giardia 2041 cytosolic fractions separated on SDS-PAGE gels (12% acrylamide) and silver stained demonstrate diverse protein profiles. M, molecular mass markers (in kilodaltons); lane 1, whole cytosolic extract; lane 2, fraction 7; lane 3, fraction 10; lane 4, fraction 12; lane 5, fraction 13; lane 6, fraction 15; lane 7, fraction 26; lane 8, fraction 27; lane 9, fraction 28; lane 10, fraction 29; lane 11, fraction 31; lane 12, fraction 33; lane 13, fraction 32; lane 14, fraction 36; lane 15, fraction 37; lane 16, fraction 39; lane 17, fraction 40.

Inoculation regimen. KLH (0.5 to 5 mg per mouse) or Flu (100 µg per mouse) antigen was prepared alone or with CT (1 or 10 µg per mouse) or with Giardia preparations. In every case the amount of Giardia extract or secreted product administered to individual mice was derived from 10⁷ trophozoites. This value is the maximum number of isolate 106 trophozoites which colonize a mouse intestine in the standard laboratory mouse infection model (32). KLH prepared in combination with each of the 12 Giardia cytosolic fraction pools or KLH alone was dialyzed against PBS for 2 h at 4°C to produce a preparation suitable for administration to mice. Subsequently only the high-salt-containing preparations (KLH plus Fx12 and KLH plus Fx7) were dialyzed.

Sample collection and processing. Preimmune fecal and blood (tail vein) samples were collected from individual mice prior to inoculation. Fecal pellets (4 to 5) and blood samples (10 to 100 µl) were routinely collected from individual mice on a weekly or biweekly basis. Blood, saliva (10 to 100 µl), small intestine (gut) washings (2 ml), and whole small intestines (guts) were collected from each mouse on experiment termination.

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View larger version (18K): [in this window] [in a new window]   FIG. 2.   Trophozoite-adjuvanted anti-KLH serum IgG titers vary relative to Giardia isolate. Groups of four Quackenbush mice were inoculated with 3 mg of KLH alone or with 107 trophozoites on days 0 and 7. Pooled serum samples were collected from each group on days 14, 21, and 28, and anti-KLH serum IgG titers were detected by ELISA.

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Antibody ELISA. Enzyme-liked immunosorbent assay (ELISA) plates were coated overnight with 2 to 5 µg ml¹ of antigen (KLH, Flu, or CT) or with a 1:100 dilution of Giardia 2041B cytosolic extract in 50 mM carbonate buffer (pH 9.0) (15 mM Na₂CO₃, 35 mM NaHCO₃, 3 mM NaN₃) at 4°C, then washed with PBS-0.05%Tween 20 (Sigma), and blocking solution containing 0.5% BSA (Sigma) in PBS was added for 1 h at RT. Plates were washed, and samples were added in dilution buffer (0.05% BSA, 0.05% Tween 20 in PBS) and serially diluted within the plate. Preimmune sera and fecal samples, and control saliva, gut washes, and gut homogenate samples collected from either Quackenbush or C57BL/6J × CBA control mice were included to determine cutoff values for antibody titers.

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Calculation of titers. Three times the background OD at 415 nm or 450 nm reading from control samples was used as a cutoff to determine antibody titers. For the Flu experiments, titers were calculated using the mean + three standard deviations of the OD₄₅₀ reading from preimmune samples at each dilution as a cutoff value. All serum titers are presented on a logarithmic scale and secretory titers on an arithmetic scale, which best demonstrate the range of titers from these extracts. All mean values have been calculated arithmetically.

	RESULTS

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Adjuvant activity of Giardia trophozoites. To assess the adjuvant activity of Giardia trophozoites, we coadministered to mice whole Giardia 2041 or 106 trophozoites with KLH antigen using a two-dose regimen and observed an increase in anti-KLH serum IgG titers over those detected for mice inoculated with KLH alone (Fig. 2). By experiment day 28, anti-KLH serum IgG titers were twofold higher for the 2041 group (20,480) compared with the 106 group (10,240) (Fig. 2). When, using the same parameters, 2041B trophozoites were coadministered with a range of KLH concentrations, anti-KLH serum IgG titers measured on experiment day 28 diminished relative to antigen dose as follows; 5,120 at 3 mg/mouse, 2,560 at 1 mg/mouse, 1,280 at 0.5 mg/mouse, and 1,280 at 0.5 mg/mouse of nonadjuvanted KLH. Increased concentrations of KLH (up to 5 mg/mouse), coadministered with Giardia trophozoites, did not appreciably increase the anti-KLH serum IgG titers (data not shown).

View larger version (60K): [in this window] [in a new window]   FIG. 3.   Total mucosal IgA measured on the intestinal mucosa of Giardia trophozoite-infected mice. Three-day-old Quackenbush mice were each inoculated p.o. with one dose of either Giardia 2041 (C) or 106 (B) trophozoites (5 × 105 per mouse) or with PBS (A) as a control. The small intestine was collected at 14 days postinfection, and IgA was bound with goat anti-mouse IgA-HRP antibody. The bar in each panel represents 100 µm.

Adjuvant activity of Giardia 2041 crude extracts. Using a freeze-thaw and sonication technique, Giardia trophozoites were fractionated into membrane and cytosolic extracts. We coadministered 2041 trophozoites, cytosolic extract, or membrane fraction with KLH antigen using a three-dose regimen. Anti-KLH serum IgG titers peaked on experiment day 28 (Fig. 4A), with titers from the cytosolic extract and trophozoite groups (20,000) being greater than from the membrane fraction (2,000) and KLH alone (6,500) groups. An anti-KLH serum IgG1 and IgG2b response was detected, while there was no measurable anti-KLH serum IgG2a or IgG3. Anti-KLH serum IgG1 peaked by day 31 and anti-KLH serum IgG2b by day 35 in the trophozoite- and cytosolic extract-adjuvanted groups (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   KLH-specific antibody titers from mice inoculated with antigen and crude Giardia extracts. Groups of six Quackenbush mice were inoculated with 5 mg of KLH alone or with Giardia extracts from 107 2041 trophozoites on days 0, 7, and 21. Serum and fecal samples were collected on days 0, 7, 31, and 35 and pooled for antibody detection by ELISA. Tr, trophozoites; Cyt, cytosolic extract; Mem, membrane extract.

Reproducibility of Giardia 2041 adjuvant activity. Our findings thus far have demonstrated that the cytosolic fraction of Giardia 2041 trophozoites, rather than the membrane fraction or secreted product, is the predominant source for adjuvant activity. We subsequently assessed the reproducibility of this activity through comparing antibody responses in mice administered antigen and different batches of 2041 cytosolic extract, measuring antibody titers from individual mice rather than from pooled groups, and comparing the level of Giardia adjuvant activity with the "gold standard" control adjuvant, CT.

Variation in Giardia adjuvant activity with antigen. To confirm that the enhanced antibody responses seen with administration of KLH and Giardia crude extracts were not confined to one antigen, we repeated our studies using influenza virus (Flu). In contrast to KLH, there was no significant increase in anti-Flu serum IgG titers in the Giardia cytosolic extract- or CT-adjuvanted groups measured 1 week after three doses (data not shown). Flu is a strong antigen in its own right, and after four doses, by experiment day 58, average anti-Flu serum IgG titers had increased in all groups, with only the CT-adjuvanted group titer (45,900) above that of the Flu-alone group (24,400).

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Giardia 2041 adjuvants a Flu-specific serum (A) and secretory (B) IgA response. Groups of 8 to 10 C57BL/6J mice were inoculated with 100 µg of Flu alone or with Giardia 2041 cytosolic extract (extracted from 107 trophozoites) or 10 µg of CT on days 0, 7, 28, and 42. On day 58, individual serum samples were collected and gut homogenates were prepared from the excised small intestine of individual mice. Samples were assayed for the presence of Flu-specific antibodies using ELISA. Results are presented for individual mice, with the average group titer represented as a bar. Cyt, cytosolic extract.

Adjuvant activity of fractions from Giardia 2041 cytosol. To further investigate Giardia adjuvant activity, 2041 cytosolic extract was fractionated, and samples of pooled fractions 1 to 12 were prepared, which were subsequently coadministered with KLH to mice using a two-dose regimen. CT was also coadministered with KLH, producing a control adjuvant group.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Giardia 2041 pooled cytosolic fractions variously adjuvant a KLH-specific serum IgG (A) and serum IgA (B) response. Groups of five Quackenbush mice were inoculated with 3 mg of KLH alone or with Giardia 2041 pooled fractions, each extracted from the equivalent of 107 trophozoites, or 1 µg of CT on days 0, 7, and 21. Individual mouse serum samples were collected on day 30 (B) and day 35 (A) for antibody analysis by ELISA. Results are presented for individual mice in each group, with the average group titer represented as a bar.

	DISCUSSION

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Cytosolic components of Giardia isolate 2041 cultured trophozoites are capable of adjuvanting an antigen-specific serum and secretory antibody response to orally coadministered antigen in a mouse model. The Giardia-adjuvanted serum antibody response was comparable to that generated by the "gold standard" mucosal adjuvant, CT. Evidence of enhanced adjuvant activity induced by isolate 2041 over isolate 106 is consistent with previous observations of Giardia toxin-like properties (36, 10). We have also confirmed an increased production of total serum IgA without production of Giardia antigen-specific IgA in 2041-infected mice (36), an ideal attribute of adjuvant activity.

Similarly, our mouse infection model demonstrates Giardia trophozoite (particularly 2041)-induced secretion of total IgA in the gut epithelium of neonatal 17-day-old mice, which normally have an immature immune system in which the processes of antibody affinity maturation and isotype switching are in general poorly achieved (19). By 3 weeks of age the neonatal immune system has matured, and IgA is secreted within the gut epithelium regardless of incitement (data not shown).

The source of adjuvant activity was localized to the trophozoite cytosol rather than the membrane fraction or secreted product of the parasite. Although some activity from the secreted product might be expected, culture conditions are far removed from the environment experienced by the parasite in an animal host and therefore may not be conducive to secretion of toxins or other products. In earlier experiments (data not shown), proteinase K digestion and 60°C heat treatment of 2041 cytosol reduced adjuvant activity, indicating that a significant portion of the adjuvant molecule is protein. When different batches of cytosolic extract were used, limited variation was seen in antigen-specific serum IgG antibody titers. However, any variability between batches may result from variation in parasite growth phase, cell count, time of harvest, and variability in processing.

Both antigen and adjuvant doses play a role in production of an immune versus a tolerogenic immunological response to inoculation. Often a large dose of antigen with a strong mucosal adjuvant is required for administration via the oral route to overcome the phenomenon of oral tolerance (12). Inoculation quantities in experimental studies are usually on the order of 100 to 5,000 µg of antigen and at least 50 µg of adjuvant (22), although smaller quantities (1 to 10 µg) of purified CT (33, 39) and nanogram concentrations of LT (13) have successfully adjuvanted a mucosal response. In keeping with these concentrations, Giardia extracts were administered using the equivalent of 10⁷ trophozoites, which equates to approximately 500 µg (data not shown) of total protein. At this stage we have not identified what portion of this value can be equated with the adjuvant factor(s). The quantity of 10⁷ trophozoites was assessed as the maximum number of trophozoites which colonize a single mouse intestine in the standard laboratory mouse infection model (32). In an earlier experiment (data not shown), mice were administered a quarter dose of 2041 cytosol and KLH antigen, which produced anti-KLH serum IgG titers at 1.5 weeks subsequent to dosing of up to 200 times less than those measured with administration of a full dose (equivalent to 10⁷ trophozoites) of cytosol.

Giardia extracts were administered with a limited range of KLH concentrations, and a lower limit of 3 to 5 mg per mouse was determined to be the most suitable concentration for production of a strong antigen-specific antibody response comparable to those recorded in the literature (33, 38). When Flu was administered at a concentration of 100 µg per mouse, significant anti-Flu serum IgG titers were detected with only the CT-adjuvanted group and not with the Giardia adjuvants. Average anti-Flu serum IgA titers were enhanced from the CT- and Giardia cytosolic extract-adjuvanted groups. Anti-Flu secretory IgA titers varied with the type of secretory extract tested (fecal, saliva, gut washing, or gut homogenate). The highest and most consistent titers were measured in the gut homogenates of the cytosolic extract-adjuvanted groups. High IgA titers in gut homogenate samples may correlate with the content of the lamina propria, which is rich in IgA plasma cells and expected to produce higher antigen-specific titers than gut washings alone. It is difficult to compare adjuvant activity, on an equivalent basis, of the crude Giardia extracts with highly purified and potent CT. Crude Giardia adjuvant activity may not be of sufficient potency to produce a secretory response in the less concentrated mucosal samples (fecal and gut washing) or at distal mucosal sites, e.g., in saliva. The concept of a common mucosal system where inoculation of a preparation at one site stimulates a response at all other mucosal sites does not always follow and may vary with the type of adjuvant administered (5).

The source of adjuvant activity from Giardia 2041 trophozoites was localized to at least one pool of cytosolic fractions, fraction pool 12. This fraction pool demonstrated the strongest adjuvant activity throughout the 3-week postinoculation period and generated adjuvant activity at a similar level or higher than the titers recorded for CT. Giardia trophozoites, cytosolic extract, and fraction pool 12 produced antigen-specific serum IgG1 and IgG2b isotypes, indicative of a Th2 response. This is in keeping with the response generated by CT and its mutants, which primarily stimulate a Th2 response through increased production of interleukin-4 (IL-4), IL-5, and IL-10, with predominant production of IgG1 and IgE (22) and in some cases IgG2b (9) isotypes. This polarization in the T-cell response, however, varies with adjuvant type and is less pronounced with LT, which activates both Th1 and Th2 cells (22).

In comparison with other Giardia extracts, and regardless of dose regimen, we were unable to detect an enhanced antigen-specific secretory response with administration of the fraction pools. The ability to adjuvant a secretory response may be diminished with Giardia protein preparation purification, causing separation of potent adjuvant complexes. This is not totally unforeseen, as loss of adjuvant activity has been recorded with the preparation of mutant molecules of CT and LT, especially those in which the toxic A1 subunit has been removed (22). If, like CT, the Giardia adjuvant extracts bind to receptors on the gut epithelia (22), an alteration in adjuvant structure might affect its binding ability, therefore preventing transport to immune inductor sites for stimulation of an immune response. It is possible that more than one form of adjuvant is contained within the Giardia trophozoites, with different forms adjuvanting the immune response via a range of mechanisms, which differentially affect the systemic and mucosal compartments. We intend to further define the Giardia adjuvant molecule(s), including its physical properties, which will allow a more direct comparison by weight with CT and other available adjuvants.

For standard prophylactic immunization in healthy individuals, only adjuvants that induce minimal side effects will prove acceptable (24). The critical issue of vaccine safety has precluded many candidate adjuvants from further development due to their toxic form. Our studies have demonstrated that extracts produced from the pathogenic Giardia 2041 isolate function with adjuvant-like activity in an animal model. The same animals do not display overt behavioral changes synonymous with a toxic response to inoculation, such as reduced mobility, loss of appetite, and substantial weight loss. These results indicate that Giardia extracts are worthy of further investigation as a future source for candidate mucosal and systemic adjuvants.