1 Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera #239, DF 07320, Mexico
2 División de Biología Molecular, Instituto Finlay, AP 16017 Ciudad de la Habana, C.P. 11600, Cuba
3 Departamento de Patología Experimental, CINVESTAV, IPN, AP 14-740, DF 07000, Mexico
4 Departamento de Biología Molecular, CENIC, AP 6690 Ciudad de La Habana, Cuba
Correspondence
Esther Orozco
esther{at}mail.cinvestav.mx
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ABSTRACT |
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INTRODUCTION |
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Two proteins have been mainly used in protection experiments: the galactose/N-acetylgalactosamine inhibitable lectin (Petri et al., 1987) and the serine-rich protein (Stanley et al., 1990
), both of which are involved in adherence. Immunization experiments using DNA plasmids carrying genes encoding these proteins have also been done and have shown that these DNAs induce an immune response to E. histolytica (Gaucher & Chadee, 2002
; Zhang & Stanley, 1999
). Immunization with DNA produces a broad-spectrum and long-lasting immune response, better cross-strain protection and a reduced cost in terms of production of the vaccine (Kowalczyk & Ertl, 1999
). In addition, the unmethylated CpG motifs in the backbone of DNA vectors elicit secretion of cytokines by lymphocytes, including IL-12 and TNF-
(Kowalczyk & Ertl, 1999
), which promote the development of the Th1 immune response (Campbell & Chadee, 1997
).
EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in E. histolytica's cytopathic effect, adhesion and phagocytosis (Arroyo & Orozco, 1987; Garcia-Rivera et al., 1999
; Rodriguez et al., 1989
). The EhCPADH complex has a molecular mass of 124·6 kDa and is located in the plasma membrane and cytoplasmic vacuoles (Garcia-Rivera et al., 1999
). Our previous results have shown that hamsters immunized with a recombinant fragment of EhCPADH (Garcia-Rivera et al., 1999
) were partially protected against liver abscesses (Martinez-Lopez et al., 2004
). Two adjacent genes encode EhCPADH (Garcia-Rivera et al., 1999
). The Ehcp112 gene encodes a 446 aa polypeptide with 2731 % identity to other cysteine proteases. EhCP112 contains a putative transmembrane sequence (aa 395416) and an integrin attachment domain (RGD) (Garcia-Rivera et al., 1999
). The Ehadh112 gene encodes a 687 aa polypeptide (EhADH112), which contains three predicted transmembrane segments (aa 114130, 176195 and 224247) and the cell-adherence domain located at the carboxy terminal region (Garcia-Rivera et al., 1999
). The monoclonal antibody (mAb) anti-EhCPADH inhibits trophozoite adherence and recognizes an epitope in the carboxy domain of EhADH112 with homology to another epitope located in the EhCP112 polypeptide (Garcia-Rivera et al., 1999
). Further characterization of the EhCPADH complex is important to better understand the functions of both of its polypeptides.
In this work, we studied the expression of EhCP112 and EhADH112 after plasmids encoding them had been used independently to transfect fibroblasts and to inoculate hamsters. Both proteins were expressed on the fibroblasts' surface and both were expressed in the animals inoculated with the plasmids, measured by their immune response against E. histolytica trophozoites.
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METHODS |
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Construction of plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 and transfection of L929 fibroblasts.
The full-length Ehcp112 gene (1338 bp) and a 1755 bp DNA fragment of the Ehadh112 gene, lacking 306 bp at the 5' end, were independently cloned in the BamHI/EcoRI and XbaI/ApaI sites of the vector pcDNA3 (Invitrogen), respectively (Fig. 1). Constructs were checked by sequencing using the dideoxynucleotide chain-termination method (Sanger et al., 1977
). The plasmids were purified by using Qiagen Giga-Plasmid columns. Five micrograms of each plasmid were incubated with 25 µg lipofectAmine (GIBCO) in a final volume of 2·4 ml during 25 min at room temperature, following the manufacturer's instructions. The mixture was added to the L929 culture cells at 70 % confluence and the plates with the cells were incubated at 37 °C in 5 % CO2 for 5 h. After that, medium was supplemented with 20 % FCS. Geneticin (GIBCO) [1 mg (ml medium)1] was added to the cells 48 h after transfection. For other experiments, the complete Ehcp112 gene and 729 bp at the carboxy terminus of the Ehadh112 gene were cloned in plasmid pRST A and recombinant proteins were produced in IPTG-induced Escherichia coli BL21(DE3) (Garcia-Rivera et al., 1999
).
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Inoculation of hamsters with plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112.
Syrian golden hamsters (Mesocricetus auratus) (male, 4 weeks old) were divided into groups of eight animals and intradermally injected at the base of the tail with 50, 100 or 200 µg of pcDNA-Ehcp112 or pcDNA-Ehadh112 or with a mixture of 50 µg of both plasmids. As a control, we injected animals with 100 µg of pcDNA3. Animals were immunized on days 0, 20 and 40, and were bled from the retro-orbital plexus on days 0, 10, 20, 30, 40 and 50. At day 57, after three inoculations, hamsters were challenged with 1·5x105 trophozoites, inoculated via the portal vein, as described below. Animals were bled and killed under anaesthesia (6·3 mg Anaesthesal ml1) (Pfizer) 1 week after challenge (day 64).
ELISA.
ELISA plates (96-well; Costar) (Engvall & Perlman, 1971) were coated with heat-fixed trophozoites (52 °C, 15 min) resuspended in PBS (104 per well) as described by Zhang & Stanley (1996)
. Then, 50 µl of the hamster sera (1 : 500) were added to the wells. Plates were incubated overnight at 4 °C, washed three times with PBS and incubated for 2 h at 37 °C with 50 µl of horseradish peroxidase (HRP)-labelled goat anti-hamster IgG (1 : 2000) (Cappel). The enzymic reaction was developed as described by Engvall & Perlman (1971)
; the colour developed was measured in an ELISA reader (ICN, MS2) at OD492.
SDS-PAGE and Western blot assays.
Trophozoites were washed twice with PBS at 4 °C and disrupted by freezingthawing in the presence of 20 mM p-hydroxymercuribenzoate (Sigma). Samples were electrophoresed on SDS-PAGE (12 % polyacrylamide) gels and transferred to nitrocellulose membranes (Towbin et al., 1979), which were blocked with 2 % non-fat milk in 0·5 % Tween 20 in PBS for 1 h. Membranes were incubated with the hamster anti-sera (1 : 100) or with the anti-EhCPADH mAb overnight at 4 °C, and then for 1 h at room temperature with HRP-labelled goat anti-hamster IgG (Cappel) or goat anti-mouse IgM (Zymed), respectively, and developed with 4-chloro-1-naphthol and 0·04 % H2O2.
In vitro cell proliferation assay.
Hamsters were immunized and challenged with live trophozoites as described above. Seven days after challenge (day 64), animals were killed and their spleens were placed in 5 ml of serum-free RPMI medium. The lymphocytes were obtained and resuspended at 2x106 (ml RPMI medium)1 supplemented with 10 % heat-inactivated FCS, 100 U penicillin ml1 and 100 µg streptomycin sulphate ml1. For T-cell proliferation, 2x105 lymphocytes per well were cultured with 1x104 or 4x104 heat-killed trophozoites (52 °C, 15 min). As a negative control, lymphocytes were incubated with serum-free RPMI medium; as a positive control, lymphocytes were incubated with 2·5 µg ml1 concanavalin A (Pharmacia) per well. Cells were cultured for 48 h at 37 °C in a humid atmosphere containing 5 % CO2. Then, 1 µCi (37 kBq) per well of [3H]thymidine (Amersham Pharmacia) was added to the cultures for an additional 18 h. Incorporated radioactivity was measured by liquid scintillation counting (Beckman Instruments LS5801) in cells washed and filtered through a glass filter paper for cell harvest (Nunc).
Adherence inhibition assays using hamster antisera.
Trophozoites (6x105) were pre-incubated at 4 °C for 20 min with 1·2 µl immune hamster sera adjusted to the same titre and diluted 1 : 500 in serum-free TYI-S-33 medium. Then, 600 µl of human red blood cells (RBCs) (1x108 cells ml1) were added and cell mixtures were incubated for 10 min at 4 °C. RBCs adhered to 100 trophozoites were counted as described previously (Garcia-Rivera et al., 1982). The adherence efficiency was calculated taking as 100 % the RBCs adhered to trophozoites that were incubated in the absence of serum. As controls, we used sera from hamsters inoculated with pcDNA3 or with the pre-immune sera.
Virulence in vivo.
Under sterile conditions and intra-peritoneal anaesthesia (6·3 mg Anaesthesal ml1), groups of eight hamsters for each condition were laparotomized and intraportally infected with 1·5x105 trophozoites in a volume of 0·1 ml PBS using a 29 gauge needle (Tsutsumi et al., 1984). Seven days after the challenge, the animals were anaesthetized, their livers were removed and the number and size of the abscesses were recorded.
Statistical analysis.
Collected data were submitted to ANOVA, and the less statistical difference (LSD) test was used to determine significant differences between the experimental groups. The probability level for significance was 95 %. All values are expressed as mean±SD.
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RESULTS |
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Plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 are expressed in the plasma membrane of mammalian cells
We investigated whether plasmids pcDNA-Ehcp112 and pcDNA-Ehadh112 were able to express in mammalian cells and the protein location in transfected cells. L929 fibroblast cells transfected with each one of the plasmids and grown in medium supplemented with neomycin were examined every day. After 4 days, all the non-transfected cells died due to the antibiotic, whereas cells transfected with either of the plasmids survived. Cells transfected with Ehcp112 (Ehcp112-L929 cells) and Ehadh112 (Ehadh112-L929 cells) presented differences. Whereas the Ehcp112-L929 cells appeared rounded and detached from the plate and many of them died, Ehadh112-L929 cells seemed to be always healthy. The appearance of the Ehcp112-L929 cells was similar to that presented by cultured cell monolayers incubated with trophozoite extracts. The effect may be due to the presence of mature EhCP112 in the cells, which is toxic for mammalian cells. One week after the experiments, the Ehcp112-L929 cells showed only 10 % survival. However, a few days later, the surviving cells divided and grew well. In contrast, 7585 % of the cells transfected with pcDNA-Ehadh112 survived. We confirmed the expression of the E. histolytica proteins in the Ehcp112-L929 and Ehadh112-L929 cells by immunofluorescence using the anti-EhCPADH mAb (Fig. 2a, b), which recognizes common epitopes in the EhCP112 and EhADH112 polypeptides. We have not precisely identified the common epitopes recognized by the mAb, but EhCP112 and EhADH112 share sequences in their C terminus region that our preliminary phage display experiments suggest are recognized by the mAb (Fig. 2
, bottom). The anti-EhCPADH mAb reacted with the surface of the Ehcp112-L929 and Ehadh112-L929 cells (Fig. 2b
). In all experiments, 99 % of the cells gave fluorescence, although intensity was higher in Ehadh112-L929 than in Ehcp112-L929 cells (Fig. 2b, a
). Fluorescence was not detected in the cells transfected with pcDNA3 (Fig. 2d
), nor in transfected cells incubated only with the secondary antibody (Fig. 2c
). These results indicated that pcDNA3 drove the expression of the cloned genes to produce the EhCP112 and EhADH112 proteins in the mammalian transfected cells. In addition, the recombinant proteins were sorted to the plasma membrane of Ehcp112-L929 and Ehadh112-L929 cells as the native proteins are in live trophozoites (Arroyo & Orozco, 1987
).
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The specificity of the humoral response was investigated by Western blot assays using total E. histolytica proteins and a sera pool from hamsters immunized three times with 100 µg of pcDNA-Ehcp112 or pcDNA-Ehadh112. In both cases, the sera reacted with the EhCPADH complex (Fig. 3c, d, lane 1), which was also recognized by the anti-EhCPADH mAb (Fig. 3c, d
, lane 2). The pre-immune sera and the sera from the animals immunized with vector pcDNA3 did not recognize any band (Fig. 3c, d
, lane 3, 4), indicating a specific humoral response against the EhCPADH protein complex. Interestingly, the sera from animals inoculated with pcDNA-Ehcp112 or pcDNA-Ehadh112 did not recognize the native EhCP112 (49 kDa) and EhADH112 (75·5 kDa) in total trophozoites proteins, suggesting that the polypeptides form the EhCPADH complex immediately after their synthesis and that the amount of free EhCP112 and EhADH112 is very low to be detected by these antibodies. As expected, the recombinant EhCP112 and EhADH112 proteins produced in bacteria were recognized by the anti-EhCPADH mAb and by the respective immune sera against EhCP112 and EhADH112 polypeptides (Fig. 3e, f
). Anti-EhCPADH mAb and the immune sera of the immunized animals with pcDNA-Ehcp112 reacted with 52 and 40 kDa proteins in the lane where the rEhCP112 protein was electrophoresed (Fig. 3e
). Fifty-two kilodaltons is the expected molecular mass of the polypeptide according to the cDNA fragment cloned in the vector, and the 40 kDa band may correspond to a degradation product of the whole recombinant EhCP112. In contrast, the anti-EhCPADH mAb and the antibodies of the immunized animals with plasmid pcDNA-Ehadh112 reacted with a 30 kDa band in the lane where is a 30 kDa recombinant fragment of the EhADH112 protein (Fig. 3f
) cloned in the expression vector pRSET B (Martinez-Lopez et al., 2004
).
pcDNA-Ehcp112 and pcDNA-Ehadh112 induce a cell-mediated immune response in hamsters
We evaluated the cellular immune response in the immunized animals, 1 week after challenging them with virulent trophozoites. Sixty-four days after the first immunization, cell proliferation was measured in spleen lymphocytes cultured in the presence of [3H]thymidine and on 1x104 or 4x104 heat-killed trophozoites per well for in vitro stimulation. No significant variations were found with the two different numbers of trophozoites used. Animals inoculated with 100 µg of pcDNA-Ehadh112 gave a better response (876911 352 c.p.m.) than those inoculated with 100 µg of pcDNA-Ehcp112 (23622581 c.p.m.). Differences between these two groups were significant in all experiments (P<0·05). The proliferative response of the control animals that were injected only with the vector and challenged with live trophozoites was 5781009 c.p.m. (Fig. 4a, b).
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DISCUSSION |
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According to the TMPRED program (Hofmann & Stoffel, 1993), the polypeptides encoded by the Ehcp112 gene and the Ehadh112 gene fragment used here have transmembrane sequences. This allowed their expression in the fibroblast plasma membrane, in a way that was analogous to native protein expression in the trophozoites. The recombinant antigens may also be exposed on the hamster cells' surface favouring the stimulation of the humoral immune response.
EhCP112 has a signal peptide, explaining its presence in the Ehcp112-L929 cells. Intriguingly, EhADH112 does not have a detectable signal peptide, which poses the question as to how this recombinant protein went to the plasma membrane in the Ehadh112-L929 cells. One possibility is that its delivery to the plasma membrane could have been achieved by a signal-peptide-independent pathway similar to other membrane or secreted proteins reported in other organisms (Cho & Cummings, 1995; Denny et al., 2000
; Gray et al., 1989
; Kim et al., 1998
; Lopez-Ordonez et al., 2001
; Rubartelli et al., 1990
; Trudel et al., 2000
; Zhao & Morales, 2000
). If this protein has a signal that allows it go to the plasma membrane, this should be situated at its C terminus, because the recombinant polypeptide used here does not contain the first 106 aa of the N terminus. There are many reports indicating that in some proteins the amino acid sequence required for membrane driving or for secretion is situated at the C terminus (Gray et al., 1989
; Kim et al., 1998
; Lopez-Ordonez et al., 2001
). However, more studies are needed to confirm this.
The EhCPADH complex induces a good humoral response both in hamsters and humans (Arroyo & Orozco, 1987; Martinez-Lopez et al., 2004
). The humoral response may account for the protection observed in the animals inoculated with the mixture of the plasmids. In fact, studies using both active and passive immunization strategies suggest that certain antibodies to E. histolytica may protect against liver abscesses (Lotter et al., 1997
; Seydel et al., 1996
; Zhang et al., 1994
). The cellular response did not augment in the protected animals, although with these experiments we cannot discard differences in the quality of the cellular response with the different immunization protocols. Humoral response produced by the independent polypeptides and by the combination of them was rather low (Figs 3a and 5a
), but specific, as was shown by the recognition of the EhCPADH protein complex by the hamster antibodies (Figs 3c, d and 5b
). The low response could be due to the low amount of antigen produced by the plasmids. In ELISA assays we used heat-killed trophozoites as antigen; perhaps a better response would be obtained using the recombinant EhCP112 and EhADH112 proteins. However, recombinant EhCP112 protein was obtained in a very low amount, probably due to its toxicity, and both recombinant polypeptides are easily degraded.
The specificity of hamsters' antibodies was confirmed by their recognition of the EhCPADH complex in Western blot assays using total trophozoite proteins and by their adherence-inhibition ability in the in vitro assays. However, the in vivo virulence of the trophozoites was not affected in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112. Our results and those of many others indicate that the complexity of in vivo virulence cannot be compared with in vitro studies.
Zhang & Stanley (1999) did not find differences in the antibody titres of vaccinated animals that were or were not protected from liver abscess. Here, we found that inoculation with both plasmids together improved the humoral response of the hamsters; however, we do not know the precise contribution of the specific antibodies to the reduction of liver damage observed in this group.
It has been reported that DNA vaccines promote the cellular response more than the humoral one (Kowalczyk & Ertl, 1999), and experimental evidence suggests that the cellular response has a predominant role in protection against amoebiasis (Campbell & Chadee, 1997
; Schain et al., 1995
), although the role of the humoral response has also been documented (Lotter et al., 1997
; Seydel et al., 1996
; Zhang et al., 1994
).
Interestingly, inoculation with the plasmid mixture improved the immune response of the hamsters and reduced both the number and the size of the hepatic abscesses observed. It is conceivable that the expression in the animals of both polypeptides may promote the formation of a protein complex similar to the native EhCPADH in trophozoites. This could favour the presentation of more representative antigenic epitopes of the native complex to the immune system, providing a more-specific stimulation for T and B lymphocytes. However, this needs to be investigated further.
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ACKNOWLEDGEMENTS |
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Received 25 November 2003;
revised 10 February 2004;
accepted 13 February 2004.
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