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Institute of Parasitology, McGill University, Macdonald Campus, 21 111 Lakeshore Rd, Ste-Anne de Bellevue, Québec H9X 3V9, Canada
Correspondence
Gaétan M. Faubert
gaetan.faubert{at}mcgill.ca
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Lactococcus lactis is amongst the best characterized of the lactic acid bacteria and has been proposed to serve as an oral mucosal vaccine delivery system (Norton et al., 1994; Robinson et al., 1997; Wells et al., 1996). L. lactis is being intensively investigated as a delivery vehicle for bacterial/viral antigens and cytokines at mucosal sites. For example, Brucella abortus L7/L12 protein (Pontes et al., 2003; Ribeiro et al., 2002), tetanus toxin fragment C of Clostridium tetani (Norton et al., 1996, 1997; Robinson et al., 1997, 2004; Wells et al., 1993), human papillomavirus-16 E7 oncoprotein (Bermudez-Humaran et al., 2004; Cortes-Perez et al., 2005) and human IL-10 (Steidler et al., 2000) have all been successfully expressed in L. lactis.
Giardia lamblia is an intestinal protozoan parasite disseminated worldwide and a major pollutant of surface water. The infection is spread by the cyst stage and as little as 10 cysts are required for infection. Giardiasis is usually acquired through drinking cyst-contaminated water or eating cyst-contaminated food. The clinical illness is characterized by diarrhoea, abdominal cramps, bloating, weight loss and malabsorption; however, asymptomatic infections also frequently occur (Faubert et al., 2002). Although giardiasis cases occur sporadically, water-borne outbreaks are well documented. During 1991–2000 in the United States, Giardia was identified as a causal agent of 9.4 % (10 of 106) of recreational-water-associated outbreaks and 16.2 % (21 of 130) of drinking-water-associated gastroenteritis of known or suspected infectious aetiology (Hlavsa et al., 2005).
G. lamblia has a direct life cycle and possesses two stages: the trophozoite, which is the vegetative stage colonizing the small intestine, and the cyst, which is released into faecal material constituting the infective stage. The cyst is composed of a rigid wall structure which protects the two trophozoites inside from the harsh external environment. Cyst wall proteins (CWPs) with a molecular mass ranging from 29 to 102 kDa have been detected by immunostaining (Erlandsen et al., 1990). One of these, CWP2, appears to be a major component of the cyst wall structure. The intestinal mucosal immune system of mice infected with Giardia muris recognizes CWP2 as a foreign antigen since specific antibodies are produced locally (Larocque et al., 2003). In addition, we have reported that immunization of BALB/c mice with the 39 kDa form of CWP2 reduces cyst shedding in the murine model of giardiasis when it is delivered orally with cholera toxin (Larocque et al., 2003). CWP2 has two forms. Newly expressed CWP2 has a molecular mass of 39 kDa and possesses a hydrophobic N-terminal signal peptide followed by five leucine-rich repeat regions and a cysteine-rich region with a 13 kDa carboxy end tail region (Lujan et al., 1995). However, mature CWP2, detected within the cyst wall, migrates at 26 kDa since it lacks its 13 kDa tail portion due to post-translational processing. Through epitope mapping, we have determined that the mature 26 kDa form of CWP2 contains the relevant B cell epitopes (P. Lee & G. Faubert, unpublished data). Therefore, we have chosen to focus on this form of CWP2 in our study.
Since lactic acid bacteria have been proposed to serve as oral mucosal vaccine delivery systems, we engineered L. lactis for cell surface expression of CWP2. The aims of this study were threefold. First, we sought to determine if L. lactis can express a parasite protein derived from G. lamblia. Second, using the nisin-inducible expression system, we would then examine the efficiency of subcellular localization of the 26 kDa mature form of CWP2 (intracellular, secreted or cell-surface-anchored) by L. lactis. Third, we would determine whether recombinant lactococci expressing CWP2 on the cell surface can successfully deliver CWP2 to the intestinal mucosal sites of mice, generating CWP2-specific IgA antibodies. Secretory antibodies against Giardia spp. play a central role in the clearance of this parasite from the intestinal tract of the host (Eckmann, 2003; Langford et al., 2002). Although IgA has been implicated in giardial clearance, there are currently no studies in the literature documenting IgA and its role with Giardia cysts. We report that L. lactis can successfully express CWP2, a protein of parasite origin, at three different subcellular locations. Moreover, lactococcal cells expressing CWP2 on the cell surface were able to stimulate CWP2-specific IgA antibodies which lead to a significant reduction in cyst shedding. In a pilot challenge experiment, mice immunized with recombinant lactococcal cells demonstrated a 63 % reduction in cyst output. This result is promising since it validates a strategy to target and control the infective stage of the parasite which warrants further investigation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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5 thi (lac-proAB) F'(traD36 proAB-lacZM15)] (Gibson, 1984) and L. lactis htrA-NZ9000 (MG1363 derivative carrying nisK and nisR genes on the chromosome, Emr, htrA disrupted by single-crossover recombination) (Le Loir et al., 2001) were used as bacterial hosts. htrA encodes an L. lactis extracellular housekeeping protease responsible for the degradation of exported and fusion proteins (Poquet et al., 2000). L. lactis htrA-NZ9000 was kindly provided by P. Langella and I. Poquet (INRA, Jouy-en-Josas, France). E. coli was grown in Luria–Bertani (LB) medium at 37 °C in an orbital shaker. L. lactis was grown without agitation at 30 °C in M17 medium (Difco) (Terzaghi & Sandine, 1975) supplemented with 1 % glucose (GM17). When solid medium was required, agar (1.5 %) was added to GM17 to make GM17 agar plates. When required, antibiotics were added as follows: for L. lactis, chloramphenicol was added at a concentration of 10 µg ml–1 and erythromycin at 5 µg ml–1; for E. coli, chloramphenicol was added at 10 µg ml–1 and erythromycin at 150 µg ml–1. Unless indicated, plasmid constructions were first established in E. coli (Sambrook et al., 1989) and then transformed into L. lactis by electroporation (Langella et al., 1993).
Plasmid engineering.
The different plasmids used in this experiment were kindly provided by P. Langella (INRA, Jouy-en-Josas, France); their characteristics are described in Table 1. All plasmids (pCYT : Nuc, pSEC : LEISS : Nuc and pVE5547 : L7/L12) were extracted from E. coli TG1 by alkali lysis and purified using a CsCl gradient. pCYT and pSEC backbones are derivatives of pNZ8010 (de Ruyter et al., 1996). pCYT : CWP2 and pSEC : LEISS : CWP2 were constructed by replacing the nuc gene with the CWP2 gene in pCYT : Nuc and pSEC : LEISS : Nuc. The constructed plasmids together with protein localization are illustrated in Fig. 1.
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). Giardia lacks introns and in encysting G. lamblia trophozoites, cyst wall proteins are transcribed as intron-free mRNAs (Lujan et al., 1995). Using different sets of primers listed in Table 2, CWP2 DNA fragments were amplified to be incorporated into the different vectors. All PCR products were resolved by agarose gel electrophoresis (0.8 %) and visualized by ethidium bromide. DNA bands of interest were purified from the gel, using a GFX PCR DNA and Gel Band Purification Kit (Pharmacia Biotech), according to the manufacturer's recommendations.
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, b). The CWP2 PCR fragment was digested with SalI and EcoRV enzymes and cloned directly into SalI/EcoRV-digested pVE5547 : L7/L12, resulting in pVE5547 : CWP2, encoding an in-frame fusion of CWP2 with the M6 cyst wall anchor sequence (CWAM6). However, pVE5547 is a large theta-replicating plasmid that is difficult to manipulate (Dieye et al., 2001). Therefore, an SPUsp-CWP2-CWAM6 cassette was transferred from pVE5547 : CWP2 into a pGK derivative, a smaller E. coli shuttle vector that is easier to manipulate (Bermudez-Humaran et al., 2002, 2003; Kok et al., 1984). Briefly, the SPUsp-CWP2-CWAM6 cassette was amplified by PCR from pVE5547 : CWP2 using primers 5 and 6 and digested with BamHI and EcoRI. The digested cassette fragment was then cloned into the pGK backbone purified from pCYT : Nuc digested with BglII and EcoRI, resulting in plasmid pVE5547CORE : CWP2. BglII was used to create a compatible end for the BamHI site of the SPUsp-CWP2-CWAM6 cassette. A pre-existing BamHI site was present elsewhere within pCYT : Nuc.
Electroporation.
Electroporation was performed as described by Holo & Nes (1995) with modifications as suggested by Geller et al. (2001). The electroporation was carried out using a Bio-Rad Gene Pulsar II Electroporator apparatus at 25 mF, 2.00 kV and a Gene Controller set at 200 
using 0.2 cm gap electroporation cuvettes. Immediately after electroporation, the cells were resuspended in M17 containing 15 % sucrose, 1 % glycine and supplemented with 20 mM MgCl2 and 2 mM CaCl2, and incubated at 30 °C for 2 h without any agitation to allow the cells to recover from the electroporation. After recovery, the cells were plated on GM17 agar plates containing antibiotics and incubated for 1–2 days at 30 °C.
Nisin induction.
L. lactis strains were grown without agitation at 30 °C overnight in GM17 medium containing antibiotics. Selected clones were inoculated in fresh GM17 at a 1 : 50 dilution and grown until early/mid-exponential phase, which corresponds to an OD600 of 0.3–0.5. Induction was carried out using different concentrations (0.1, 1 or 10 ng ml–1) of nisin (Sigma). After induction, cells were grown without agitation at 30 °C for 1–3 h before performing protein extraction from the induced cells.
Growth of recombinant lactococcal cells in the presence of nisin.
Overnight cultures of L. lactis strains were diluted 1 : 50 in fresh GM17 medium containing antibiotics. The bacteria were grown to an OD600 of 0.4 and induced with 10 ng nisin ml–1, which was previously determined to be the optimal amount of nisin for CWP2 expression. Subsequently, the OD600 of all cultures was measured prior to induction and subsequently every half hour for up to 7 h after induction using a Biochrom Ultraspec 2000 Pro UV/Visible Light spectrophotometer. Samples (1 ml) were collected from each bacterial strain under sterile conditions to produce a 7 h growth curve.
Protein extraction.
Protein extracts were prepared from exponentially growing cultures as described by Le Loir et al. (1998). For cell fractionation, 10 ml L. lactis culture was centrifuged for 5 min at 6000 g at 4 °C and both the supernatant and cell pellet fractions were collected for immunoblot analysis.
Immunoblotting.
To determine if CWP2 was produced after nisin induction of the vector promoter, immunoblotting was performed. L. lactis lysate samples were boiled for 10 min in an equal volume of reducing sample buffer and applied in volumes of 25 µl. Proteins were separated on a 4 % stacking gel and 12 % separating gel using a Bio-Rad Protean II electrophoresis unit. Resolved proteins were transferred onto a nitrocellulose membrane using a mini-gel Bio-Rad transfer apparatus set at 100 V for 1 h at 4 °C. Non-specific binding sites on the blotted nitrocellulose membrane were blocked with TBS containing 1 % Tween-20 (TTBS) and 5 % skim milk overnight. The nitrocellulose membrane was then washed with TTBS and incubated for 1.5 h at room temperature with mAb 8C5.C11 diluted 1 : 1500, or rabbit polyclonal 10F5 antibody diluted 1 : 3500. The polyclonal antibody 10F5 is specific against M6 (SIGA), while mAb 8C5.C11 is specific against CWP2 (Campbell & Faubert, 1994a). Membranes were then washed with TTBS and subsequently incubated with a goat anti-mouse horseradish-peroxidase-conjugated secondary antibody (1 : 3000; Amersham Pharmacia) or with a goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (1 : 5000; Cedar Lane Labs) for 1 h. Probed proteins were visualized by chemiluminescence using Super Signal West Pico Chemiluminescent Substrate (Pierce), according to the manufacturer's recommendations. They were subsequently exposed to a Kodak Scientific Imaging X-OMAT AR film for 5–10 min and developed in a Kodak M35A Film Processor.
Immunofluorescence.
To confirm the presence of the CWP2-M6 fusion protein on the cell surface of recombinant lactococci, cells were examined by immunofluorescence as described by Cortes-Perez et al. (2003). Briefly, 5 ml nisin-induced L. lactis cells were harvested at an OD600 of 0.5–0.6, corresponding to mid-exponential phase. The cells were incubated overnight at room temperature with mAb 8C5.C11 (1 : 1500), specific against CWP2, diluted in PBS/1 % BSA. After three washes with PBS/Tween 0.05 %, the immune complex was incubated for 6 h at room temperature with a goat anti-mouse IgG conjugated to fluorescein isothiocyanate diluted 1 : 100. Cells were washed three times. Smears were prepared, air-dried and heat-fixed. A cover slip was added to the slides and mounted using Vectashield mounting medium (Vector Labs). The slides were examined under UV illumination using a Nikon Eclipse 800 compound microscope at x100 magnification with an FITC filter (502 nm).
Preparation of L. lactis for immunization.
Selected clones were inoculated in fresh GM17 at 1 : 50 dilution and grown until early/mid-exponential phase, corresponding to an OD600 of 0.3–0.4. Lactococcal cell pellets were harvested by centrifugation at 3000 g for 15 min at 4 °C. Cells were washed three times with sterile PBS and were subsequently resuspended in GM17 (no antibiotics) to a final concentration of 2.5x1010 c.f.u. ml–1.
Immunization of BALB/c mice.
To assess the immunogenicity of recombinant lactococcal cells, 26 BALB/c mice were divided into four groups and immunized by oral gavage. The first group (n=7) was not immunized and served as a negative control. The second group (n=6) was immunized with htrA-NZ9000 which served as a bacterial control group. The third group (n=6) was immunized with lactococcal cells expressing M6 protein (C-terminal half of the molecule) fused to B. abortus L7/L12 antigen. This group served as a control for M6 protein being expressed on the cell surface. The last group of mice (n=7) received lactococcal cells carrying pVE5547CORE : CWP2 and expressing CWP2-M6 fusion protein on the cell surface. Every mouse received 1010 c.f.u. bacteria per dose in a volume of 0.4 ml GM17 medium. Mice received six doses within a 30 day period (days 1, 2, 3, 15, 16 and 17).
Collection of gut lavage fluids.
Gut lavage fluids were obtained by flushing the excised small intestine with 5 ml PBS containing 50 mM EDTA and 1 % BSA (Boehringer Mannheim) as described by Wu & Russell (1993). Subsequently, lavage samples were vortexed and centrifuged at 1000 g for 15 min at 4 °C. Supernatants were removed and 50 µl 100 mM PMSF (Sigma) was added to the supernatants before they were vortexed and spun at 5000 g for 20 min at 4 °C. Supernatants were dispensed into aliquots and frozen at –20 °C. until further use.
CWP2-specific antibody responses.
Anti-CWP2 IgA antibodies in intestinal lavage fluids were measured by ELISA as described by Medaglini et al. (2001). Flat-bottom microtitre ELISA plates (Falcon) were coated with 100 µl G. lamblia encysting cell antigen at a concentration of 1 µg ml–1 in PBS and blocked with PBS/1 % albumin. Encysting cell antigen was prepared as described by Larocque et al. (2003) and contains native CWP2 expressed by G. lamblia. Intestinal lavage samples were diluted 1 : 2 followed by twofold dilutions. Plates were incubated at room temperature for 1 h. After washing, 100 µl anti-mouse IgA diluted 1 : 10 000 (Caltag Laboratories) conjugated to horseradish peroxidase was added to the wells. After the plates were incubated for 1 h at room temperature, the substrate 3,3',5,5'-tetramethylbenzidine (Sigma) was added to the wells. Plates were read at 450 nm after 20 min using an EL309 Microplate Reader (Bio-Tek Instruments). Results are expressed as the ratio of the amount of CWP2 specific antibodies (µg) to the amount of total antibody (mg) in the sample for IgA. The amount of CWP2-specific antibodies cannot be compared alone between groups since the amount of IgA in intestinal lavage fluids may vary from mouse to mouse which may influence the amount of specific antibodies. As such, the amount of CWP2-specific antibodies was normalized to the total amount of antibodies detected within the sample in the form of a ratio to compare groups.
Challenge of BALB/c mice with G. muris cysts.
Twelve BALB/c mice were divided into four groups of three mice each. Mice were immunized as described above. Thirteen days after the last dose (day 30), all mice were challenged by gavage without anaesthesia with 6x105 live G. muris cysts. The Giardia mouse model of infection was selected for two reasons: first, mice can be challenged with G. muris cysts and second, cross-reactivity exists between G. muris and G. lamblia cyst antigens. Campbell & Faubert (1994a) were able to stain both G. lamblia and G. muris cysts using mAb 8C5.C11, thus indicating the existence of a G. muris CWP2 murine homologue containing conserved epitopes. Cyst output was followed for all mice for a period of 15 days post-challenge. No adverse signs were noted in any mice and no mice died during challenge with G. muris.
Isolation of cysts from faecal specimens.
For a period of 15 days (days 6–20 post-challenge), individual mice were placed in separate cages and the faecal pellets excreted over a 1 h period were collected in 12x75 mm glass borosilicate tubes. Cysts were isolated by a sucrose gradient centrifugation technique as described previously (Campbell & Faubert, 1994b). Briefly, faeces were collected, weighed, emulsified in PBS, layered on sucrose (specific gravity 1.12) and centrifuged at 400 g for 15 min. Cysts were counted with a Spencer Bright Line haemocytometer (Fisher Scientific).
Statistics.
Statistical significance was determined by Student's unpaired t-test performed using SigmaStat 3.11 statistical analysis software from SYSTAT Software Inc. The significance level was set at P
0.001.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, with the addition of nisin at a concentration of 1 or 10 ng ml–1, CWP2 was expressed (Fig. 2a–c). With the Western blot assay, a band at 
26 kDa was detected using CWP2-specific mAb 8C5.C11 for lactococcal cells harbouring vector pCYT expressing CWP2 (Fig. 2a) or vector pSEC : LEISS expressing CWP2 (Fig. 2b), regardless of the amount of nisin used for induction. This band at 26 kDa corresponds to the molecular mass of CWP2. In addition, when lactococcal cells were transformed with vector pVE5547CORE and induced with 1 or 10 ng nisin ml–1, a band at the 52 kDa mark was detected (Fig. 2c). The increase in molecular mass of CWP2 is due to pVE5547CORE expressing CWP2 as a fusion protein with M6 (26 kDa), therefore doubling its molecular mass. Using rabbit polyclonal antibodies specific against the M6 anchoring protein, a similar band of 52 kDa was detected, confirming that CWP2 is expressed as a CWP2-M6 fusion protein when vector pVE5547CORE is used (data not shown).
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). Vectors pSEC : LEISS (Fig. 3b) and pVE5547CORE (Fig. 3c) secreted CWP2 into the supernatant and expressed it on the cell surface, respectively. A band at 26 kDa appeared when CWP2 was expressed in the cytosol or secreted into the culture medium (Fig. 3b). When CWP2 was anchored to the cell surface, a band appeared at 52 kDa due to the fusion of CWP2 with M6 (Fig. 3c).
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), but did stain positive with mAb 10F5 (Fig. 4c). On the other hand, cells engineered with vector pVE5547 : CORE, expressing CWP2 as a fusion protein with M6, stained positive with both mAbs (Fig. 4b and d).
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). The mean ratio of the CWP2 group was fivefold higher than the non-infected control mice [203.93±24.31 µg (mg total IgA)–1 versus 41.07±8.80 µg (mg total IgA)–1]. All mice (7 out of 7) within the CWP2 group were positive for the presence of CWP2-specific intestinal IgA using the mean of the values of the non-infected control mice plus 2 SEM as a cut-off (dotted line). No significant levels of CWP2-specific IgA were observed with the other groups, thus indicating that lactococcal cells by themselves (NZ9000), the presence of the Streptococcus pyogenes M6 anchor protein or the B. abortus L7/L12 protein (L7/L12) do not induce antibodies which cross-react with the CWP2 antigen. The NZ9000 group had a mean ratio of 95.13±22.66 µg (mg total IgA)–1 whereas the L7/L12 group had a mean ratio of 69.40±17.60 µg (mg total IgA)–1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gilbert et al., 2000; Miyoshi et al., 2002). We report, for the first time, on the expression of an intestinal parasite protein by L. lactis. This protein originates from the protozoan parasite G. lamblia which is a major pollutant of surface water (Faubert et al., 2002). CWP2 is an important building block of the cyst wall which protects the parasite against the harsh external environment (Lujan et al., 1995). Using the appropriate vector and the optimal concentration of nisin, L. lactis can have CWP2 expressed intracellularly, secreted into culture medium or anchored to the cell surface. In addition, we report that intragastric immunization of BALB/c mice with recombinant lactococcal cells was able to deliver CWP2 to the mucosal site. In a pilot experiment, mice immunized with recombinant lactococci expressing CWP2 on the cell surface and challenged with live Giardia cysts shed a significantly lower number of cysts.
L. lactis has been used to express successfully proteins from infectious agents and a variety of cytokines (Nouaille et al., 2003). This study with CWP2 is only the second report in the literature documenting the expression of a protein of parasitic origin. The other study described a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein and the C-terminal portion of the merozoite surface protein 3 of Plasmodium falciparum which was successfully expressed and secreted by L. lactis (Theisen et al., 2004). However, in that study, L. lactis was used as an expression system, not as a live vaccine delivery system.
CWP2 is considered to be one of the major proteins involved in the composition of the cyst wall, a structure allowing G. lamblia to survive outside of its host in the environment. CWP2 is generally not secreted into the medium when expressed by the parasite, but it can be found at the cell surface within the cyst wall. Using vector pVE5547CORE together with nisin as an inducing agent, CWP2 was expressed by L. lactis and anchored to the cell surface, mimicking how CWP2 is normally expressed by the parasite. This form of expression may have important implications for vaccine development since the immune system normally sees CWP2 in this context (i.e. displayed on the cell surface). CWP2 was also expressed in the cytosol of L. lactis or secreted into the culture medium. We did not observe any advantage in having CWP2 expressed in any one particular form with respect to growth rates. No significant differences in growth rates were seen between lactococcal cells harbouring different vectors when CWP2 expression was induced. These findings reinforce the versatility of L. lactis as an expression vehicle for foreign proteins, since L. lactis can be targeted to multiple subcellular locations using nisin-inducible expression system vectors (de Vos, 1999; Miyoshi et al., 2002; Nouaille et al., 2003).
Although we demonstrated that CWP2 can be expressed at different subcellular locations, we were predominantly interested in having CWP2 anchored to the cell surface for the purpose of developing an oral vaccine. To minimize proteolysis of CWP2 when expressed on the cell surface, we utilized L. lactis htrA-NZ9000, a mutant strain where the htrA gene has been inactivated. htrA encodes an extracellular housekeeping protease responsible for the degradation of exported and fusion proteins (Frees et al., 2001; Nilsson et al., 1994; Poquet et al., 2000). To confirm this, Western blotting revealed one major predominant CWP2 band when L. lactis htrA-NZ9000 was employed, whereas multiple CWP2 bands were detected with L. lactis NZ9000 (data not shown). The presence of multiple bands were indicative of CWP2 being degraded, supporting the idea that an htrA-deficient L. lactis strain provides higher protein stability at the cell surface (Miyoshi et al., 2002).
Previous studies comparing different forms of antigen expression and subsequent immune responses have indicated that cell-surface expression results in a better immune response than intracellular or secreted forms of antigen presentation (Bermudez-Humaran et al., 2004; Cortes-Perez et al., 2003). It is believed that the bacterial cell wall may provide an adjuvant activity, enhancing the host immunological response (Vitini et al., 2000). In addition, cell-surface-displayed antigens are less soluble than their secretable counterparts in most cases (Bernasconi et al., 2002; Lindholm et al., 2004) and may be less exposed to degrading or denaturing agents, such as proteinases, or acid-rich environments such as the stomach (Nouaille et al., 2003; Piard et al., 1997a, b). Lactococcal cells expressing CWP2 on the cell surface were immunogenic, generating a significant amount of CWP2-specific secretory IgA antibodies detected from intestinal lavage samples of mice immunized with recombinant lactococci. Giardiasis in humans and in mice results in the production of antigiardial antibodies of the IgA and IgM isotypes in mucosal secretions and IgG in serum (Faubert et al., 2002). Moreover, specific antibody production in mucosal sites correlates with giardial clearance (Daniels & Belosevic, 1994; Heyworth, 1992, 1986; Heyworth et al., 1987; Nash et al., 1987; Snider & Underdown, 1986). However, the physiological role of IgA and IgM in clearing Giardia infection from the intestine is unknown.
Mice immunized with recombinant lactococcal cells expressing CWP2 on the cell surface released 63 % less cysts per gram faeces than non-immunized control mice. Our observed reduction level compares quite well with the 75 % reduction level demonstrated from a previous study where mice were orally immunized with the full-length 39 kDa form of recombinant CWP2 produced by E. coli (Larocque et al., 2003). Since both stages of the Giardia life cycle occur simultaneously in the intestine (Campbell & Faubert, 1994b), the immune clearance of the cysts from the intestinal lumen may be due to antibodies directed at the trophozoite stage. However, the delivery of CWP2 to the intestinal mucosa of mice by engineered lactococcal cells gave rise to cyst-specific antibodies only. No trophozoite-specific antibodies were detected at the intestinal mucosal site. Moreover, the number of trophozoites was compared between the different groups of mice (data not shown). No differences were observed between the CWP2-immunized and the non-immunized group, indicating that the trophozoite population was not targeted by the immune response. Thus, reduction of cyst shedding is not due to the immune elimination of the trophozoite stage, but due to the antibodies directed against CWP2, which is a major component of the cyst wall structure, thereby inhibiting the formation of the cyst structure.
Experiments are in progress to elucidate the immune mechanisms involved.
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ACKNOWLEDGEMENTS |
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Received 29 January 2006;
revised 8 March 2006;
accepted 15 March 2006.
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