Giardia lamblia infection induces different secretory and systemic antibody responses in mice

C. VELAZQUEZ¹, M. BELTRAN², N. ONTIVEROS², L. RASCON¹, D. C. FIGUEROA¹, A. J. GRANADOS¹, J. HERNANDEZ-MARTINEZ³, J. HERNANDEZ² & H. ASTIAZARAN-GARCIA²

SUMMARY

The adult mouse model of Giardia lamblia infection serves as an excellent animal model to understand the immunological mechanisms involved in the control and clearance of Giardia infection. Little is known about the G. lamblia-specific antigens that stimulate the humoral immune response in this model of giardiasis. We analysed the secretory and systemic antibody responses to G. lamblia during primary and secondary infection in C3H/HeJ adult mice. Faecal IgA and Serum IgG anti-G. lamblia antibodies were observed at week 2 post-infection. Serum IgG responses remained constant over the next several weeks, whereas faecal IgA titres continued to rise from weeks 2–6 post-infection. Western blot analysis revealed that intestinal IgA and serum IgG antibody responses were directed toward several distinct proteins of G. lamblia. Certain proteins appeared to be recognized by both faecal IgA and serum IgG, whereas other antigens were specific for either the secretory or systemic antibody responses. G. lamblia primary and secondary infections were associated with differences in the antibody recognition pattern. The biochemical and immunological characterization of these antigens will help us to better understand the immunobiology of the G. lamblia–host interaction.

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The protozoan parasite Giardia lamblia is an intestinal parasite of humans, which the World Health Organization estimates to have infected 250 million people worldwide (1). Clinical manifestations of G. lamblia infections vary from the asymptomatic carrier state to severe diarrhoea, abdominal pain, nausea, malabsorption and weight loss (2,3). Giardia infections are usually self-limiting in immunocompetent individuals, indicating the presence of effective host defence mechanisms against this intestinal parasite (2–5). Despite the high incidence and clinical importance of G. lamblia infections, the immunological mechanisms that play a role in giardiasis are poorly understood.

The mouse model of giardiasis is a powerful tool to study the immune effector mechanisms that occur during Giardia infections, and has considerable advantages over other animal models (6–9). The immune system of the mouse is well characterized, an extensive variety of reagents and technologies exist for the study of the mouse immune system, and immunologically well-defined inbred strains of mice are available. Byrd et al. reported the development of a G. lamblia–adult mouse model that uses a well-characterized clone of G. lamblia (GS/M-83-H7) (6,10). The adult mouse model of G. lamblia infection has been used to better understand the immunological mechanisms active in giardiasis as well as the antigenic variation in G. lamblia (5,10–14). Additionally, the mouse model has helped to gain new insight into the key elements of the immune response that play a role in giardiasis. Several studies have shown that T and B lymphocytes are important to control Giardia infection in mice (5,10,11,14). However, our knowledge about the specific antigens of G. lamblia, which induce a humoral and cellular immune response, remains limited.

In the present study, we evaluated the secretory and systemic antibody response during the course of a primary and secondary G. lamblia infection in C3H/HeJ adult mice. Immunogenic antigens of G. lamblia were identified that induce mucosal and serum antibody responses. Our data demonstrate differences in antigen recognition between secretory and systemic antibody responses, and have implications for future research on giardiasis.

C3H/HeJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). This strain of mice is susceptible to infection with the G. lamblia clone GS/M-83-H7 (11). Trophozoites from the G. lamblia clone GS/M-83-H7 were obtained from the American Type Culture collection (ATCC 50581). Axenic G. lamblia cultures were maintained in the TYI-S-33 medium with antibiotics.

Soluble G. lamblia trophozoite antigens were obtained by using the method described by Gottstein et al. (15) with slight modifications. Briefly, G. lamblia trophozoites from confluent cultures were harvested during log-phase by chilling on ice for 30 min. One hundred million trophozoites were washed three times with sterile phosphate buffer saline (PBS), resuspended in 1·5 mL of PBS, frozen (liquid nitrogen) and thawed (room temperature) three times, and then sonicated (30 cycles for 2 min [Brandon sonifier 250, Shelton, CT, USA] in the presence of protease inhibitor cocktail [23 mM/L 4-(2-aminoethyl) benzenesulphonyl fluoride (AEBSF)], 0·3 mM/L pepstatin A, 0·3 mM/L E-64, 2 mM/L bestatin, and 100 mM/L sodium EDTA [Sigma, St. Louis, MO, USA]). Cell debris was removed by centrifugation (10 000 g for 30 min). The protein concentration of the soluble antigen preparation was determined by the Bradford method (Bio-Rad, Hercules, CA, USA).

Eight- to 10-week-old male C3H/HeJ mice were infected with 5 × 10⁶ trophozoites of the G. lamblia clone GS/M-83-H7 by using a sterile animal feeding needle for peroral inoculation. The G. lamblia inoculum was prepared by washing in vitro cultivated trophozoites three times with ice-cold sterile PBS and resuspending them in 200 µL of sterile PBS. Primary infection occurred on day 0, with secondary challenge taking place on day 45.

To evaluate the antibody responses during a primary and secondary G. lamblia infection in mice, we did at least four different experiments (at least four animals/experiment). The animal experiments shown in the paper are representative experiments and are consistent with all the experiments performed during this study.

Blood and faecal sampling of mice began (day 0) prior to G. lamblia infection and was performed weekly for 6 weeks after primary infection and again for 6 weeks after secondary challenge. Mice were bled from the tail vein and serum was recovered and stored at −30°C. To analyse the intestinal anti-G. lamblia (IgA) antibodies, faecal extracts were prepared as follows: two to three faecal pellets of each mouse were collected in a microcentrifuge tube containing 0·5 mL of PBS-1% bovine serum albumin (Sigma) and 1 mM phenylmethanesulfonyl fluoride (PMSF). Tubes were incubated overnight at 4°C. The suspension was vortexed vigorously for 10 s and centrifuged at 10 000 g at 4°C for 10 min to remove insoluble material. Supernatant was collected and stored at −30°C (12).

Total faecal IgA was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates (Corning, Corning, NY, USA) were coated overnight at 4°C with 125 ng (50 µL) of sheep anti-mouse IgA (α-chain specific) (Sigma). Wells were blocked with PBS containing 1% BSA (PBS-1% BSA) for 1 h at room temperature. After washing, 50 µL of faecal extracts (diluted 1 : 100 in PBS-1% BSA) were added to triplicate wells and incubated for 90 min at room temperature. After washing, 50 µL of peroxidase goat anti-mouse IgA (Zymed, San Francisco, CA, USA) diluted 1 : 1000 in PBS-1% BSA was added to each well for 1 h at room temperature. Plates were washed and developed with 1 mm 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABT-S; Boehringer Mannheim GmbH, Mannheim, Germany) in citrate buffer with 0·03% H₂O₂. Optical density (at 415 nm) was read at 30 min with an ELISA reader (Benchmark Microplate Reader, Bio-Rad, Hercules, CA, USA). For each ELISA plate, a standard curve was constructed using purified mouse IgA (Sigma).

To evaluate serum anti-G. lamblia IgG and intestinal anti-G. lamblia IgA of infected mice, an ELISA was carried out using standard techniques. Briefly, 96-well plates (Corning) were coated with 50 µL (2·5 µg) of soluble G. lamblia antigen in 0·1 M sodium bicarbonate buffer pH 9·6. After overnight incubation at 4°C, plates were washed with PBS-0·05% Tween 20 (PBST), and blocked with PBS-1% BSA for 1 h at room temperature and washed. Faecal extracts (15 ng of total intestinal IgA) and mouse serum samples (diluted 1 : 10 in PBS-1% BSA) from both infected and non-infected mice were added to triplicate wells and incubated for 1 h at room temperature. After washing with PBST, antibody binding was detected with 50 µL of HRP-conjugated goat anti-mouse IgG (1 : 1000 diluted in PBS-1% BSA) (Sigma) or HRP-conjugated goat anti-mouse IgA (1 : 1000 diluted in PBS-1% BSA) (Zymed, San Francisco, CA, USA). After 1 h of incubation for IgG ELISA plates and 90 min of incubation for IgA ELISA plates (both at room temperature), the plates were washed, and developed with 1 mm ABT-S in citrate buffer with 0·03% H₂O₂. Optical density was measured at 415 nm. Statistical analysis was performed using paired t-tests.

Soluble G. lamblia proteins were separated by SDS-PAGE (12%) under reducing conditions without boiling and subsequently stained with Coomassie Brilliant Blue (Bio-Rad) by standard methods.

In order to evaluate antibody recognition by Western blotting, 500 µg of soluble G. lamblia protein was mixed with an equal volume of 2X SDS-PAGE sample buffer (4% SDS, 2% 2-mercaptoethanol, 0·125 M Tris-HCl/0·1% SDS, 20% glycerol, and 0·001% bromophenol blue) and loaded on a preparative SDS-PAGE mini-gel (12% separating gel with a 4% stacking gel). Proteins were electrotransferred to a nitrocellulose membrane for 30 min using a semi-dry blotting system (The W.E.P. Company, Seattle, WA, USA) with 120 mAmp current. Nitrocellulose membranes were blocked with PBS containing 1% dry milk and 1% BSA for 1 h at room temperature. Blocked membranes were incubated with serum (diluted 1 : 25 with PBS-1% BSA) for 1 h at room temperature. After five washes with PBST, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (diluted 1 : 5000 with PBS-1% BSA) for 1 h at room temperature. Membranes were washed and developed using a SuperSignal West Pico Chemoluminescent kit (Pierce, Rockford, IL, USA). To evaluate the antibody recognition of faecal extracts (intestinal IgA), we used a modified 2X SDS-PAGE sample buffer (0·2% SDS, 0·2% 2-mercaptoethanol, 0·125 M Tris-HCl/0·1% SDS, 20% glycerol and 0·001% bromophenol blue) to run the soluble G. lamblia proteins in SDS-PAGE. These modifications in the SDS-PAGE sample buffer were performed after preliminary experiments revealed that standard concentration of SDS and 2-mercaptoethanol abolished the intestinal IgA antibody recognition. Peroxidase-goat anti-mouse IgA diluted 1 : 10 000 in PBS-1% BSA was used as secondary antibody, with nitrocellulose membranes developed as described previously.

In order to evaluate the course of G. lamblia infection in C3H/HeJ mice, we inoculated a group of animals (n = 12) with 5 × 10⁶G. lamblia trophozoites, and the intestinal parasite loads were evaluated at different times post-infection (p.i.). G. lamblia infection occurred in all inoculated mice. The highest parasite loads were observed between days 14 (2·5 × 10⁵ ± 1·07 × 10⁵) and 21 p.i. (1·8 × 10⁵ ± 0·24 × 10⁵). At 28 day p.i., parasites were usually undetectable. These observations are in agreement with previous reports that have shown that G. lamblia infection is controlled at 3–4 weeks p.i. in immunocompetent mice (11,14).

We used an ELISA assay to measure the anti-G. lamblia antibody response in serum (IgG) and faecal extracts (IgA) during the course of primary and secondary G. lamblia infection. A group of seven C3H/HeJ mice was infected with 5 × 10⁶G. lamblia trophozoites, and the anti-G. lamblia antibody response was evaluated at weeks 0, 1, 2, 3, 4, 5 and 6 p.i. Figure 1 shows that both systemic (Figure 1a) and mucosal (Figure 1b) anti-G. lamblia antibody responses became evident at week 2 p.i. For 3–4 weeks thereafter, constant levels of serum IgG antibody were found in all of the infected mice. In contrast, secretory IgA responses continued to rise from weeks 2–6 p.i. None of the serum and faecal samples obtained from pre-infected or uninfected animals had antibodies against G. lamblia antigens. To evaluate the antibody response during a second challenge of G. lamblia, mice were reinfected at day 45 p.i. (at this time of post-infection, the C3H/HeJ mice had cleared the G. lamblia infection). The antibody response was evaluated at weeks 0, 1, 2, 3, 4, 5 and 6 post-reinfection. All the reinfected mice showed increased levels of serum IgG and faecal IgA antibodies anti-G. lamblia (data not shown).

In order to identify the G. lamblia antigens recognized by the mucosal and systemic antibody responses of infected mice, Western blots were performed with sera and faecal extracts. Serum IgG antibody responses during a primary infection were most directed against the antigenic bands of 63 and 71 kDa (Figure 2a). This antibody reactivity was strongly detected from week 3–6 p.i. The band of 71 kDa was the first band recognized, beginning at week 2 p.i. Additional bands (159 kDa and 131 kDa) were faintly recognized from weeks 3–6 p.i. Control sera from pre-infected and uninfected mice did not exhibit any immunoreactivity. Sera collected from re-infected mice mainly recognized bands of 48, 55, 63 and 71 kDa (Figure 2b).

When the intestinal anti-G. lamblia IgA response was evaluated, considerable difficulty was encountered in optimizing the Western blotting conditions. Eventually, results were obtained by reducing the SDS and 2-mercaptoethanol concentrations in the sample buffer prior to SDS-PAGE. To evaluate the antibody recognition of faecal extracts (intestinal IgA), we used a modified 2X SDS-PAGE sample buffer (0·2% SDS, 0·2% 2-mercaptoethanol, 0·125 M Tris-HCl/0·1% SDS, 20% glycerol, and 0·001% bromophenol blue) to run the soluble G. lamblia proteins in the SDS-PAGE. These modifications in the SDS-PAGE sample buffer were performed after preliminary experiments revealed that standard concentration of SDS and 2-mercaptoethanol abolished the intestinal IgA antibody recognition. In contrast, serum IgG antibody recognition was not significantly affected by the use of standard sample buffer. Additionally, there were no appreciable differences in the electrophoretic pattern of the soluble G. lamblia proteins when diluted with modified or standard sample buffer (data not shown). The intestinal IgA antibody response during primary and secondary G. lamblia infections is shown in Figure 2 (c,d), respectively. Western blot analysis revealed that IgA antibodies in the faecal extracts reacted mainly against the bands of 63, 71, 86 and 159 kDa (Figure 2c). This antibody recognition was clearly detected from weeks 4–6 p.i. Intestinal IgA from reinfected mice recognized the 63, 71 and 86 kDa antigens from week 1 post-reinfection to the end of the experiment (Figure 2d). The antigenic band of 159 kDa was recognized from weeks 2–6 p.r.i. An additional band of 106 kDa was faintly recognized from weeks 5–6 p.r.i. Control faecal extract from preinfected and uninfected mice exhibited no immunoreactivity.

In the present study, we have identified immunoreactive antigens of G. lamblia recognized by intestinal IgA and serum IgG antibodies during the course of primary and secondary G. lamblia infection in C3H/HeJ adult mice. Infected C3H/HeJ mice showed a significant increase in the levels of intestinal IgA and serum IgG antibodies specific to G. lamblia during the period of time in which intestinal trophozoite loads began to drop considerably, suggesting that the antibody response plays an important role in the clearance of G. lamblia infection. Giardia infections in humans and mice induce the production of different classes of antibodies (IgM, IgA, and IgG) and these correlates with the clearance of infection (4,11,16). Additionally, B-cell-deficient and IgA-deficient mice were unable to control Giardia infection, indicating a central role for B cells and IgA in host defence against this parasite (11). In contrast, another study indicated that antibodies are not required for the control of acute Giardia infections (14). Additional studies need to be conducted to define the precise role of the antibody response in the adult mouse G. lamblia infection model.

Based on the amounts of total IgG and total IgA antibodies used in the respective ELISA plates and in the ELISA data shown in this paper, we can suggest that the relative abundance of anti-G. lamblia IgA antibodies in faecal extracts is much higher than serum IgG anti-G. lamblia antibodies. G. lamblia, a luminal pathogen, resides strictly in the lumen of the small intestine and does not invade through the mucosa, therefore allowing better stimulation of the mucosal immune system rather than the peripheral immune system (4). The differences in the relative abundance of specific anti-G. lamblia antibodies could be because of antigen availability to induce both local and systemic antibody responses.

Western blot analysis of faecal extracts and sera from infected and reinfected mice showed that the intestinal IgA and serum IgG antibody responses were directed to a limited number of protein bands (8), with molecular weights of 48, 55, 63, 71, 86, 106, 131 and 159 kDa. In the neonatal mouse model of giardiasis, using G. lamblia clone GS/M-83-H7, it has been reported that the serum and secretory antibody responses are almost exclusively directed against the well-characterized variable surface protein H7 (VSP H7) (15,17,18). The predicted molecular weight of this protein is 56 832 Da, but the migration of this protein in the SDS-PAGE is considerably higher (about 72 kDa). This migration of the VSP H7 varies with the acrylamide gel preparation (18). In the present study, we used the G. lamblia clone GS/M-83-H7, which expresses the VSP H7. This protein may be one of the main immunoreactive bands (i.e. band of 71 kDa) detected in the Western blotting assays. Further studies are required to characterize at the biochemical and molecular level these immunogenic antigens. The G. lamblia genome database (19), together with digestion and mass spectrometry analysis of the immunogenic antigens may help to identify these G. lamblia antigens (1).

There were several differences in the antibody recognition of the secretory and systemic antibody responses. The immunogenic band of 86 kDa was intensively and exclusively recognized by the intestinal IgA antibody response. In contrast, the proteins of 48 and 55 kDa were only recognized by the serum IgG antibody of reinfected animals. On the other hand, the bands of 63 and 71 kDa were detected by both intestinal IgA and serum IgG antibody responses. The observed differences between the secretory and systemic antibody responses could be because of several parasite and host factors. These include the nature and immunogenic capacity of the G. lamblia antigens to induce an immune response, antigen availability to induce both local and systemic antibody responses, antigen handling by antigen presenting cells which could affect the antigen's access to the systemic immune system, as well as the well-known anatomic and functional differences between the mucosal and peripheral immune systems (20–22).

G. lamblia primary and secondary infections were associated with differences in the antibody recognition pattern. The main bands recognized by the secretory and systemic antibody responses during primary infection were the proteins of 63 kDa, 71 kDa and 86 kDa; however, during a secondary infection additional proteins were detected (bands of 48 kDa, 55 kDa, 106 kDa and 159 kDa). These changes in antibody recognition may be a consequence of the antigenic variation of the parasite. Several studies have shown that the immune response to a G. lamblia infection is influenced by the capability of the parasite to continuously change its surface antigens (13,15,23,24). We plan to evaluate the role of specific anti-G. lamblia antibodies in protection against G. lamblia infection.

In order to evaluate IgA antibody recognition by Western blotting, we modified the standard sample buffer used in our SDS-PAGE. The modified sample buffer had reduced SDS (20-fold less) and 2-mercaptoethanol (10-fold less) concentrations. IgA antibody recognition was only detected when G. lamblia proteins were diluted with modified sample buffer previous to the SDS-PAGE. This observation indicates that the denaturing and reducing conditions during SDS-PAGE completely abolished recognition, suggesting the presence of conformational epitopes present in the G. lamblia antigens, which are important for IgA antibody recognition. Serum IgG antibody recognition did not change under 'mild' or 'standard' SDS-PAGE conditions.

In summary, we have identified several immunogenic antigens of G. lamblia in the C3H/HeJ adult mouse model of giardiasis. The biochemical and immunological characterization of these proteins will provide a better understanding of the G. lamblia–host interaction, and will be important for developing effective control strategies against giardiasis.