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(The FASEB Journal. 2003;17:621-627.)
© 2003 FASEB

Specific antibody immune response against the parasitic portion of a glutathione-S-transferase fusion protein

ARACELY LÓPEZ-MONTEON, ANGEL RAMOS-LIGONIO, LETICIA PÉREZ-CASTILLO, PATRICIA TALAMÁS-ROHANA and JOSÉ LUIS ROSALES-ENCINA1

Experimental Pathology Department, CINVESTAV-IPN, México DF, México

1Correspondence: Departamento de Patología Experimental, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, Del. Gustavo A. Madero, México, D.F., México. CP 07360. E-mail: rosales{at}mail.cinvestav.mx (J.L. Rosales-Encina).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The humoral immune response against an Entamoeba histolytica recombinant protein has been investigated. The 628 bp Bam HI-Eco RI DNA fragment (L1b) from the M11 cDNA clone, partially coding for a 220 kDa (L220) protein, was ligated in-frame into the pGEX-3X plasmid vector to produce the fusion protein GST-L1b. BALB/c mice were immunized with different doses of the GST-L1b fusion protein (10–500 µg). GST-L1b doses of 100/50/50, 300, or 500 µg induced an antibody response (IgG1>IgG3, IgG2a>IgG2b) specific for the amoebic part of the fusion protein (L1b). These antibodies were able to recognize the native protein in amoebic total extract. Anti-GST antibodies were not detected. On the other hand, doses of 10/10/10 or 200/100/100 µg induced antibodies able to recognize both GST (IgG2a>IgG1>IgG2b) and L1b (IgG1, IgG2a>IgG3>IgG2b). When mice were immunized with GST alone (100/50/50, 300 or 500 µg), antibodies against GST-L1b or GST were not detected. However, GST doses of 10/10/10 or 200/100/100 µg induced an antibody response able to recognize both GST-L1b and GST. We propose that an immunization protocol similar to the one used in this work may allow induction of high antibody titers specific against the parasite segment of a GST-fusion protein.—López-Monteon, A., Ramos-Ligonio, A., Pérez-Castillo, L., Talamás-Rohana, P., Rosales-Encina, J. L. Specific antibody immune response against the parasitic portion of a glutathione-S-transferase fusion protein.


Key Words: Immunization • antibodies • isotype • induction • humoral response


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANTIBODIES HAVE PROVED to be extremely useful reagents in biomedical research. They have been used for identification and cloning of new genes, purification of proteins, immunohistochemical localization of proteins, etc. Therefore, in most cases the method of choice to raise antibodies against a specific protein is to immunize with the purified protein (1) .

The ability to obtain unrestricted quantities of a cloned protein via expression in a bacterial, fungal, or eukaryotic cell expression system facilitates the structural, functional, and immunological characterization of that protein. The rapidly increasing availability of diverse expression vectors and complementary cellular systems will eventually allow for the heterologous expression of any protein (2 , 3) . Recombinant production constitutes one of the main strategies for the generation of antigens. Gene fusion strategies are frequently used to furnish the recombinant immunogen with protein "tags," which may offer improved production (4 5 6) , simplified purification (7 , 8) , or even increased immunogenicity (9 10 11 12) . Recombinant technology can thus provide strategies for efficient production and purification of antigens to be further analyzed in immune response studies. However, some problems are often found when expressing fusion proteins, such as solubility and stability of the expressed protein during the purification procedure and the inability to remove the carrier protein due to the inaccessibility of the furnished cleavage site. Removing the protein tag is important if the aim is to study the antibody immune response against the antigen expressed as a fusion protein (13) . Glutathione-S-transferase (GST) fusion systems include vectors that codify for either a thrombin cleavage site (14) or a factor Xa cleavage site (15) . However, the enzymatic digestion of fusion proteins is sensitive to different parameters (temperature, pH, ionic strength, buffer composition, etc); if the fusion protein is not properly folded, the cleavage site is not accessible (13) . However, due to the problems mentioned above, it is necessary to develop other strategies to obtain a specific antibody response. A selection of humoral immunity (antibody production) or cellular immunity can be obtained depending on the route of primary immunization, the adjuvant used, the form of the antigen, and, very important, the antigen dosage (16) .

Previously we reported that an immunogenic 220 kDa membrane protein (L220) isolated from E. histolytica (17) and L220-derived peptides were able to respectively suppress or induce T cell proliferation of spleen and lymph node cells from immunized mice (18) . In the present study we selected as a model antigen a cDNA clone (L1b) encoding for the carboxyl-terminal part of L220, which was found in the previous work to be the part responsible of inducing T cell proliferation. L1b was expressed in a bacterial system (GST fusion proteins), thus allowing the possibility for analyzing in more detail the immunological properties of the L220 antigen. Our results indicate that GST-L1b induces a highly and specific humoral immune response against the amoebic portion of the fusion protein and that this response can be modulated varying the antigen dose used in the immunization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid constructions
The BamHI-EcoRI DNA fragment (628 bp) from clone M11 (18) was subcloned in-frame in the expression vector pGEX-3X (Pharmacia, Piscataway, NJ, USA) to obtain the plasmid pGEX-L1b. The plasmid pGEX-C13, which codifies for an E. histolytica collagen binding protein, was also included (19) .

Production and purification of recombinant immunogens
Escherichia coli DH5{alpha} cells transformed with either parental or recombinant expression vectors were inoculated into LB media containing 100 mg/L ampicillin (Sigma, St. Louis, MO, USA), and incubated at 37°C overnight. Fresh LB media was incubated at 37°C with the overnight culture (1:100) and protein production was induced by addition of isopropyl ß-thiogalactoside to a final concentration of 0.1 mM. After 2 h incubation cells were harvested, and purification of expressed proteins was performed as outlined by Smith and Johnson (15) . Pelleted cells were resuspended in phosphate-buffered saline (PBS) and lysed by sonication. Lysate was then centrifuged at 10,000 rpm for 20 min and the supernatants containing solubilized fusion proteins were mixed with glutathione-agarose beads (sulfur linkage; Sigma). After adsorption for 30 min, beads were collected and washed by centrifugation. Either GST or fusion proteins were eluted by competition with free glutathione (15 mM glutathione in 50 mM Tris-HCl pH 8.0), then acetone-precipitated.

Immunization of mice
Female BALB/c mice (6- to 8-wk-old) were immunized by intraperitoneal route. All mice were maintained according to the recommendations by our Institutional Animal Care and Use Committee. Five different immunization protocols were performed 1) three doses with 10 µg of antigen, 2) one dose with 100 µg of antigen and two more with 50 µg, 3) one dose with 200 µg of antigen and two more with 100 µg, 4) one dose with 300 µg, and 5) one dose with 500 µg. First immunizations were performed with the antigen emulsified in complete Freund’s adjuvant (CFA), and reimmunizations at 1 wk intervals were performed with incomplete Freund’s adjuvant (Gibco-BRL, Grand Island, NY). The same schedule was used for control group, which received only GST plus adjuvant. At the end of the immunization scheme, animals were bled to obtain immune sera.

SDS-PAGE and immunoblotting
Proteins were resolved on 10% SDS-PAGE (20) and visualized by staining with Coomassie blue or electrophoretically transferred onto nitrocellulose paper for immunoblotting (21 , 22) . Pooled sera from each group of immunized mice were used as primary antibodies at 1:100 dilution in TBS-T (150 mM NaCl, 0.05% Tween 20, 2% skim milk, and 10 mM Tris-HCl pH 7.4). Bound antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL, USA) diluted at 1:5000, then developed with NBT and BCIP (Sigma).

Analysis of IgG subclasses
ELISA plates were coated overnight at 4°C with 2 µg/mL of GST or GST-L1b, in carbonate buffer (pH 9.6). The plates were washed six times with PBS containing 0.1% Tween (PBST) and incubated for 2 h at 37°C with blocking solution (PBS containing 5% skim milk). Plates were then washed three times with PBST, three times with PBS, and incubated with 50 µL of either mouse anti-GST (1:50) or 50 µL of anti-GST-L1b immune sera. For isotype analysis, bound antibodies were detected with affinity-purified biotinylated anti-mouse immunoglobulins (IgG1, IgG3, IgG2a, and IgG2b) antibodies (Zymed Laboratories, San Francisco, CA, USA), at 1:1000 dilution in PBST, and incubated for 2 h at room temperature. Plates were washed three times with PBST and incubated with 100 µL of a 1:1000 dilution of horseradish peroxidase-streptavidin (Zymed) for 2 h at room temperature. Plates were washed as described above, then developed with 2,2-azino-bis[3-ethylbenzthiazoline]-6-sulfonic acid (Zymed), the reaction was allowed to proceed for 20 min at room temperature. Absorbance was read at 405 nm in an ELISA reader (Labsystem Multiskan MS). For detection by Western Blot, both proteins (GST-L1b and GST) were resolved on 10% SDS-PAGE and electrophoretically transferred onto nitrocellulose paper for immunoblotting. Blots were incubated with biotinylated anti-mouse IgG1, IgG3, IgG2a, and IgG2b (Zymed) antibodies at 1:1000 dilution, detected using alkaline phosphatase-streptavidin (1:1000) (Sigma), and developed with NBT and BCIP (Sigma). To quantify the intensity of each band, a specific computer software was used (GeneSnap Version 3.00.15 and GeneTools Version 3.00.12, Synoptics, Cambridge, UK).

Statistical analysis
All the experiments reported in this paper were repeated three times using five mice per group. Pooled sera from each group of immunized mice were used in ELISA and Western blot assays. The statistical significance was determined by multifactorial ANOVA using Tukey test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and purification of fusion protein GST-L1b
Five DNA fragments were obtained from the cDNA clone M11 encoding a partial sequence of L220 (Fig. 1 ). The 628 bp DNA fragment (L1b) was subcloned in the correct reading frame and orientation into the expression vector pGEX-3X. After purification by affinity chromatography and analysis by PAGE of GST-L1b and GST, results revealed the expression of a novel protein of ~50 kDa (Fig. 2 A, lane 1) and expression of GST as a 27 kDa protein (Fig. 2A , lane 2).



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  		Figure 1. Restriction map of clone M11.



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  		Figure 2. GST-L1b and GST purification. GST-L1b (1) and GST (2) were purified from E. coli DH5{alpha} transformed with pGEX-L1b and pGEX-3X respectively. A) Coomassie blue staining and B) anti-L220 Western blot of affinity purified proteins.

The Mr of the recombinant protein L1b was identical to the theoretically predicted; its identity was confirmed by immunoblotting with anti-L220 antiserum. Anti-L220 antibodies recognized the 50 kDa GST-L1b protein together with a band of lower molecular size, likely a protein degradation product (Fig. 2B , lane 1). These antibodies showed binding only to the fusion protein and not to the GST carrier protein (Fig. 2B , lane 2).

Humoral immune response to GST-L1b
GST-L1b fusion protein was highly immunogenic (Fig. 3 A), inducing antibodies able to recognize GST-L1b up to 1:210000 dilution (data not shown), and native L220 present in amoebic total extract (ATE) up to 1:50000 dilution (Fig. 3B ) when mice were immunized with 10/10/10 µg. Sera from mice immunized with different doses of GST-L1b (Fig. 4A ) and GST (Fig. 4B ) were assayed by Western blot against purified antigens. When mice were immunized with 10/10/10 µg (a) and 200/100/100 µg (c), the humoral immune response was very strong against GST-L1b fusion protein (lane 1) and GST carrier protein (lane 2). However, when mice were immunized with 100/50/50 µg (b) and single doses of either 300 µg (d) or 500 µg (e) of GST-L1b, the humoral response was specific for the amoebic portion of the fusion protein (Fig. 4A, b, d, and e , lane 1). Preimmune serum (Fig. 4A, B, f ) did not react with GST-L1b (lane 1) or GST (lane 2) at 1:100 dilution. To confirm whether the induced antibodies were specific against the amoebic portion of the fusion protein, sera from mice immunized with different doses of GST-L1b were tested against GST-C13, a different amoebic fusion protein (Fig. 5 , lane 1), and against amoebic total extract (Fig. 5 , lane 2). Results showed that the immune sera obtained after immunization with 100/50/50 µg (b), 300 µg (d), or 500 µg (e) only recognized native L220 present in amoebic total extract (ATE); anti-GST antibodies were not detected using GST (Fig. 4 , lane 2) or GST-C13 (Fig. 5 lane 1). Moreover, sera from mice immunized with doses of 10/10/10 µg and 200/100/100 µg recognized both proteins. The results obtained with sera from mice immunized with GST-L1b and GST are summarized in Table 1 .



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  		Figure 3. Titration of anti-GST-L1b antiserum. Serum from mice immunized with 10/10/10 µg of GST-L1b was tested by Western blot assay at different dilutions against A) GST-L1b and B) amoebic total extract. (1) 1:1000; (2) 1:5000; (3) 1:10000; (4) 1:15000; (5) 1: 20000; (6) 1:30000; (7) 1:40000; (8) 1:50000, and (9) 1:80000 dilutions. (PI) preimmune serum (1:100). (L220) anti-GST-L1b antiserum against L220 protein (positive control).



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  		Figure 4. Reactivity of anti-GST-L1b and anti-GST antisera. GST-L1b (1) and GST (2) were assayed with sera (1:100) from mice immunized with different doses (µg) of A) GST-L1b and B) GST. a) 10/10/10; b) 100/50/50; c) 200/100/100; d) 300, and e) 500 µg; f) preimmune serum. Differences in the reactivity patterns seen with GST-L1b are probably due to its partial degradation depending on the purification batch used.



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  		Figure 5. Reactivity of anti-GST-L1b antiserum against the fusion protein GST-C13, and amoebic components. GST-C13 protein (1) and amoebic total extract (2) were assayed with antisera from mice immunized with different doses (µg) of GST-L1b (see Fig, 4 ). f) corresponding proteins separated by 10% SDS-PAGE stained with Coomassie blue.


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  		Table 1. Reactivity of anti-GST and anti-GST-L1b antisera induced by different immunization schemes against GST-L1b, GST, GST-C13, and amoebic total extract (ATE)

Determination of IgG subclasses of GST-L1b-specific antibodies
As can be seen in Fig. 6 , the response pattern between animals immunized with 10/10/10 µg and 200/100/100 µg of GST-L1b appears to be similar, with a predominance of IgG1, IgG3, and IgG2a isotypes (Fig. 6A ). However, when sera of these mice were assayed against GST, low level of anti-GST antibodies was detected (Fig. 6B ). Moreover, when mice were immunized with single doses of 300 or 500 µg of GST-L1b, the main isotypes produced were IgG3 and IgG1 (Fig. 6A , P <0.05). Consistently, the isotypes that recognized 6xHis-M11 fusion protein were IgG3, and IgG1 (data not shown). However, isotype recognition by ELISA was rather weak; therefore, the isotype pattern was confirmed by Western blot. The densitometry analysis of the two upper bands in the case of GST-L1b and of the main band in the case of GST, showed that with immunizations schemes using multiple doses of 10/10/10 µg (a) and 200/100/100 µg (c), the main isotypes that recognized GST-L1b (A') and GST (B') were IgG1 and IgG2a (lanes 1 and 3, respectively, P<0.05). However, in sera from mice immunized with 100/50/50 µg (b), the main isotype that recognized GST-L1b was IgG1 (A', lane 1), without recognizing GST (B', lane 1). On the other hand, in sera from mice immunized with 300 µg (d) and 500 µg (e), the main isotypes that recognized GST-L1b were IgG1 and IgG3 (P<0.05). When mice were immunized with a single dose of 500 µg of GST-L1b (e), antibodies against GST were not detected (B'), whereas a single dose of 300 µg of GST-L1b (d) induced recognition of GST mainly by IgG3 isotype (B', lane 2). This recognition may be due to the fact that in the determination of IgG subclasses the reaction is more specific.



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  		Figure 6. Isotype distribution of specific antibodies against the proteins GST-L1b (A, A') and GST (B, B'), induced by immunization with different doses of the fusion protein GST-L1b. Sera from animals immunized with different doses (µg) of GST-L1b, were assayed by ELISA (A, B) and Western blot (A', B') at 1:100 dilution, incubated with 1 ) anti-IgG1, 2 ) anti-IgG3, 3 ) anti-IgG2a, and 4 ) anti-IgG2b. a) 10/10/10, b) 100/50/50 (c), 200/100/100, d) 300, and e) 500 µg and PtdIns (preimmune serum). Values represent the mean ± SD of 5 mice per group. The two upper bands were quantified by scanning densitometry analysis. Statistical significance was analyzed by the Tukey test (P<0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using recombinant DNA technology, we studied the humoral immune response against an amoebic recombinant protein. We describe a procedure to obtain high titer and specific antibodies against the parasite portion of a GST-fusion protein. For our study, we selected the 220 kDa surface glycoprotein because it participates in the adhesion process to the host cells (23) and is a highly immunogenic molecule capable of generating high antibody titer (17) . The L1b sequence from the amoebic lectin protein (L220) was genetically fused to GST obtaining up to 20 µg/mL of culture, a yield described for other proteins using this expression system (24) .

GST-L1b fusion protein was used at different doses as the primary immunogen, since it has been suggested that a systemic immune response may be induced by administration of large doses of antigen (25) . However, this protein was highly immunogenic even with doses of 10 µg; this ability may be due to complement components, which participate after the first administration of an antigen when a primary immune response is initiated. If the GST-L1b fusion protein can interact with complement components and, as soon as antibodies appear, immune complexes are formed, small proportions of these are trapped on the surface of follicular dendritic cells. These antigenic complexes can be retained for a long period on the surface of cells and can be used for periodical restimulation of memory B cells, which are important for maintenance of the antibody response (26) . Additional experiments are needed in order to confirm this alternative.

The immunogenicity of an antigen-adjuvant formulation in terms of humoral and cellular immune responses is dependent on a range of different factors. Among these are the physical characteristics of the antigen: particulate or monomeric, charge, and degree of glycosylation, etc. It has been shown recently that not only the physical and biological activities of an antigen influence its immunogenicity, but also the dose of the antigen, can modulate the immune responses (27) . The studies described here support the proposition that the dose of antigen is crucial in determining the nature of the immune response. Several studies suggest that priming with high doses of an immunogen will induce humoral responses, whereas lower doses may lead to cell-mediated immunity (28 29 30) . However, other studies have demonstrated the opposite, depending mainly on the type of antigen used (31 , 32) .

In most studies in which whole parasites are used as immunogens, low doses can induce Th1-like responses (33 , 34) . On the contrary, when soluble proteins are used as immunogens, as the one used in this work, low doses tend to skew the response toward a Th2 type whereas a high dose induces a Th1 type. However, a super high dose of these soluble antigens turns back the immune response toward Th2 type (35) . To obtain specific humoral response against the amoebic portion of the fusion protein GST-L1b, several immunizations schemes were assayed in the murine model. The results showed that immunization with low doses of antigen induced a very strong response against both portions of the recombinant protein, but when mice were immunized with high doses of antigen the production of antibodies was specific against the parasitic portion of the fusion protein; simultaneous responses to both portions of the GST-L1b were rarely seen. However, modulation of the immunization schemes in terms of antigen dosage, route of administration, or immunization timing might increase the proportion of double responders. Our results agree with those obtained by Varley et al., who found specific responses when different adjuvants were used (36) . A possible explanation for the dose-dependent response could be the differential susceptibility of Th1 and Th2 cells to undergo apoptosis in the presence of high doses of antigen. For example, doses of 300 or 500 µg of GST-L1b could promote the outgrowth of the Th2 cells (37) , since these doses mainly induce IgG1. An additional element is the differential ability of antigen presenting cells (APC) to capture and process antigens, which is dependent on the initial form of the antigen (38) . All these factors make it difficult to establish a correlation between dose and ligand density.

Others have reported that the immune response to GST-fusion proteins can be profoundly influenced by the carrier portion of the protein and the adjuvant used (36 , 39) . This latter can be selected to suit a particular protein and to modulate the immune response in a desired direction, as to emphasize an antibody response dominated by certain isotypes or subclasses (27) . These results may play a role in the variable results obtained with GST-L1b administered in Freund’s adjuvant used in this study. We decided to determine whether the differences seen between the different protocols in our studies could be explained by the pattern of isotypes produced. As we have shown by Western blot assay, immunization of BALB/c mice with 100/50/50, 300, or 500 µg doses of GST-L1b in FA resulted in L1b-specific antibodies with a broad isotype distribution (IgG1>IgG3>IgG2a), since anti-GST antibodies were not detected.

Similar findings have been described for complete microorganisms as well as for other GST-fusion proteins. Immunization with GST-PYC2 (carboxy-terminal region of the merozoite surface protein-1) in CFA induced predominantly IgG1 (40) . Using formalin-inactivated Mycoplasma agalactiae as immunogen, Avramidis et al. reported that CFA induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes (41) . These reports demonstrate that not only GST-fusion protein, but also complete microorganisms, may induce an IgG1 response when they are administered with CFA. Similar to Al (OH)3, water-oil emulsions with immunomodulators such as mycobacteria, as in the case of CFA, predominantly induce antibodies of the IgG1 isotype in mice (42) . On the other hand, the different sensitivity among ELISA and Western blot assays could be due to the fact that the CFA adjuvant is denaturing the antigen during the emulsification process (43) ; therefore, lineal epitopes are probably not recognized by ELISA where the sample is in native conditions. For this reason, water-oil emulsions are not well suited for antigens where preservation of conformational epitopes is vital. Water-oil formulations are highly efficient for inducing T cell responses and antibody responses to linear B cell epitopes (27) .

In this work we have shown that antibody response to GST-L1b fusion protein can be profoundly altered by the dose and by the presence of antigen fused to it (36) . Finally, we propose that an immunization protocol similar to the one used in this work will allow us to obtain specific and high antibody titers against the parasite portion of a GST-fusion protein; however, this will require an empirical approach for any particular antigen.


   ACKNOWLEDGMENTS
 
We would like to thank Amelia Rios, Biol. Lidia Baylón-Pacheco, Amelia Angel-Martínez, and Enrique Martínez-de Luna for their technical assistance. This work was supported by the Consejo Nacional de Ciencia y Tecnología, México (CONACyT grants 28077M and 3480M). A.L.M. and A.R.L. were recipients of a fellowship from CONACyT.

Received for publication May 20, 2002. Accepted for publication December 20, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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