Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu, 862, CEP 04023-062, São Paulo, Brazil1
Divisão de Desenvolvimento Tecnológico e Produção, Instituto Butantan, Avenida Vital Brasil 1500, CEP 05503-900, São Paulo, Brazil2
Author for correspondence: Cátia M. Pereira. Tel: +55 11 5084 3213. Fax: +55 11 571 6504. e-mail: pereiracm{at}hotmail.com
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ABSTRACT |
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Keywords: ETEC, ST, attenuated Salmonella, flagellin fusions, mucosal vaccines
Abbreviations: ETEC; enterotoxigenic Escherichia coli; OPD, o-phenylenediamine dihydrochloride
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INTRODUCTION |
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Several lines of evidence have indicated that protection against ETEC diarrhoea involves anti-toxin and anti-bacterial components. ST is a non-immunogenic low-molecular-mass peptide (2 kDa); however, its conjugation to different carriers can lead to the induction of neutralizing antibodies. Recently, a number of efforts have been made to render ST immunogenic, including chemical coupling and genetic fusions to appropriate carrier proteins (Clements, 1990 ; Houghten et al., 1984
, 1985
; Klipstein et al., 1982
, 1983
; Sanchez et al., 1986
, 1988
). In general, chemical coupling reduced ST-associated toxicity as a function of the cross-linking and maintained the immunological determinants of ST. Genetic fusions have, as an advantage, the possibility of being delivered by live attenuated bacterial strains to elicit antibody response on the mucosal surfaces. Fusion between ST and LT resulted in a non-toxic and immunogenic molecule, capable of eliciting neutralizing antibodies against ST (Clements, 1990
). Moreover, sera and mucosal secretions from mice immunized orally with an attenuated Salmonella strain carrying this genetic fusion were able to neutralize the biological activity of native ST in the suckling mouse assay, but, surprisingly, in the absence of detectable ELISA antibody titres against ST (Cárdenas & Clements, 1993
). The OmpC outer-membrane protein of E. coli has also been used as a carrier for ST, and antibody responses to ST and OmpC proteins were elicited in rabbits immunized subcutaneously with whole cells expressing the hybrid protein; however, these antibodies were not able to neutralize the ST toxic activity (Saarilahti et al., 1989
).
Attenuated Salmonella represents an attractive vector for the delivery of heterologous antigens to the immune system. Salmonella can be rendered avirulent, for example, by the inactivation of genes involved in the biosynthesis of aromatic compounds. A number of antigens have been expressed by these attenuated strains in attempts to construct bivalent vaccines. Heterologous epitopes have also been expressed as fusions to the Salmonella flagellin, a system developed to expose epitopes on the bacterial surface, as part of the flagellar filament (Newton et al., 1989 , 1991
, 1995
; Wu et al., 1989
). Attenuated bacterial strains have also been used as carriers for DNA delivery in vivo, because of their ability to deliver the antigen-encoding DNA specifically to antigen-presenting cells at inductive sites of immune response (Darji et al., 1997
; Paglia et al., 1998
; Sizemore et al., 1995
, 1997
).
In this study, the flagellin system was used to present ST sequences to the immune system in order to obtain ST neutralizing antibodies. A sequence with 19 amino acids derived from the native ST toxin and an identical sequence except for two mutations that replace two cysteine residues by alanines were fused to flagellin, and delivered intraperitoneally as purified protein, or orally by attenuated Salmonella strains expressing the fusion protein or carrying a eukaryotic expression vector containing the fusion. The ability of these forms of presentation of the ST sequences to the immune system to elicit an immune response against ST is presented.
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METHODS |
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Cloning the flagellin gene into the eukaryotic expression vector pCDNA3.
For the construction of the eukaryotic expression vector pCDNA3 carrying the hybrid flagellin gene, a 1·8 kb EcoRISau3AI fragment containing the flagellin gene with the STmt sequence was isolated from plasmid pLS408/STmt, and was inserted in the EcoRI and BamHI sites of vector pCDNA3. The resulting plasmid, pCDNA3/FLASTmt, was introduced into the Salmonella typhimurium strain LB5000 and transferred to S. typhimurium strain SL3261.
Detection of the hybrid flagellins.
Flagellin, purified by acid cleavage as described by Ibrahim et al. (1985) , and bacterial cell lysates were separated on 10% SDS-PAGE gels. The proteins were transferred to nitrocellulose filters (Hybond-C Extra; Amersham) at 1 A for 2 h using the buffer conditions described by Towbin et al. (1979)
. The membranes were blocked with 5% low-fat milk in PBS for 1 h at room temperature, and incubated for 1 h with rabbit antiserum against Salmonella flagellar antigen d (Difco). After washings with 0·05% Tween 20 in PBS (PBS-T), bound antibodies were reacted with goat anti-rabbit IgG conjugated with peroxidase (Sigma). After incubation for 1 h with the conjugate and washings in PBS-T, bound antibodies were detected using the ECL detection system (Amersham).
Immunizations.
BALB/c mice, 68 weeks old, were immunized intraperitoneally with 10 µg purified flagellin added to 500 µg aluminium hydroxide as adjuvant on days 0, 21 and 35. Sera samples were obtained 28 d after the initial immunization and 7 d after the last booster. For oral immunization, 1010 c.f.u. of live S. typhimurium resuspended in 0·2 M NaHCO3 were fed to 812-week-old mice on days 0, 21 and 35, and fecal and sera samples from immunized animals were obtained 7 d after the second and last doses. These groups of mice received one additional dose each on day 65, and fecal and sera samples were collected 7 d after final inoculation. Groups of BALB/c mice, 68 weeks old, were also immunized in the leg muscle with four doses of 100 µg CsCl-purified plasmid pCDNA3/FLASTmt, diluted in 50 µl PBS, at 14 d intervals. Serum samples were obtained 7 d after the second, third and fourth doses.
Analyses of serum antibodies (ELISA).
Microplates (Nunc) were coated with 100 ng purified flagellin per well and incubated at 4 °C overnight. The wells were washed three times with PBS-T and blocked with 5% low-fat milk for 1 h at 37 °C. Different dilutions of mice sera were added and the plates were incubated for 1 h at 37 °C. Washes with PBS-T were performed, anti-mouse IgGperoxidase conjugate was added and incubation was continued for 1 h at 37 °C, prior to development with OPD (o-phenylenediamine dihydrochloride) and H2O2 as enzyme substrate. Detection of antibodies against ST was performed as described by Svennerholm et al. (1986) . Alternatively, microplates were coated with the ganglioside GM1 (Sigma) (1 mg ml-1) diluted in PBS and incubated at 4 °C overnight. The wells were blocked with 0·1% BSA-PBS for 30 min at room temperature. ST coupled to the purified ß subunit of cholera toxin (STCTB) (a gift from Dr A.-M. Svennerholm, Göteborg University, Sweden), 500 ng ST ml-1, diluted in PBS was added and incubation was continued for 1 h at room temperature. Different dilutions of mice antisera were added and plates were incubated for 1 h at 37 °C. Washes with PBS-T were performed, anti-mouse IgG conjugate was added and incubation was continued for 1 h at 37 °C, prior to development with OPD and H2O2 as enzyme substrate.
Inhibition ELISA.
Sera from mice immunized with purified flagellin were pre-incubated with supernatants of overnight cultures, in Casamino acids/yeast extract (CAYE), of ETEC strain PB176 (STa), or of E. coli K-12 C600 as a negative control, and added to microtitre plate wells previously coated with GM1 (1 mg ml-1) and STCTB conjugate. Washes with PBS were performed and bound antibodies were reacted with goat anti-mouse IgG conjugated with peroxidase and developed with OPD and H2O2.
Neutralization of the ST toxic activity.
The suckling mouse assay for ST was performed as described by Giannella (1976) . Mice (13 d old) were inoculated intragastrically with 0·1 ml CAYE supernatant of ETEC strain PB176 previously incubated with sera from immunized mice, for 1 h at 37 °C. After 3 h inoculation, mice were killed, the intestines were removed and the ratios between the weights of the guts and of the remaining carcasses were calculated. Gut/carcass ratios of
0·082 were considered positive.
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RESULTS |
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In order to verify the expression of the chimeric flagellin encoded by the plasmid pCDNA3/FLASTmt, mice were initially immunized intramuscularly with the purified plasmid DNA. Antibodies against flagellin were detected in sera from mice immunized with four doses of DNA (Table 2). No antibody response was detected against ST. The anti-flagellin ELISA titre obtained was significantly lower than those obtained with intraperitoneal injections of the purified flagellin. This titre difference may explain the absence of detectable antibodies against ST in mice immunized with pCDNA3/FLASTmt intramuscularly. However, these results indicated that the antigen was being expressed in eukaryotic cells, and that the construct could be employed with the attenuated live Salmonella.
Mice were then fed with live S. typhimurium carrying pCDNA3/FLASTmt and the sera of these animals were tested for the presence of antibodies against flagellin. Because of the high anti-LPS antibodies generated by immunizations with intact bacterial cells, sera from immunized mice were tested in Western blot assays with purified flagellin to avoid mistaken interpretations given by the ELISA assays using flagellin preparations which could contain some LPS contaminants. An antibody response against flagellin was detected in sera from these immunized mice, as revealed by Western blotting (Fig. 4). These sera tested negative for reactivity against ST in ELISA (data not shown). This was expected given the low titres obtained for flagellin. However, these results indicate that this alternative route of oral delivery of flagellin seems to overcome the inherent lack of antigenicity of flagellin when presented by bacteria in oral immunizations.
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DISCUSSION |
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The absence of an immune response against ST in mice immunized orally with Salmonella expressing FLAST reflects the non-immunogenic nature of flagellin through this route. Previous data have shown that serum and mucosal antibody responses against flagellin and the foreign epitope could be obtained following oral immunization with live attenuated Salmonella (Wu et al., 1989 ). Recently, however, extensive work on the Salmonella/flagellin vaccine system presented evidence that flagellin does not represent an efficient carrier for heterologous epitopes by the oral route when delivered by live recombinant attenuated Salmonella strains (Almeida et al., 1999
).
An alternative approach employed in this study to overcome the non-immunogenic nature of flagellin through oral immunization was to obtain the expression of flagellin inside the mammalian cells. Attenuated Salmonella strains have been shown to be able to deliver plasmid DNA inside eukaryotic cells and expression of the antigen in the cytosol of macrophages and dendritic cells induced the stimulation of different arms of the immune system (Darji et al., 1997 ; Sizemore et al., 1997
). Here, we showed that a systemic antibody response against flagellin could be elicited in mice orally immunized with attenuated Salmonella delivering the plasmid encoding the flagellin gene with the mutated ST to mammalian cells. Because the titres against flagellin were relatively low, we did not expect to detect an antibody response against the ST toxin. Nevertheless, this seems to be a promising method to elicit an appropriate response against fusions to flagellin. Enhancement of the specific mucosal IgA immune response by expression of interleukin-5 by an attenuated Salmonella strain has been recently demonstrated (Whittle et al., 1997
). Moreover, co-administration of interleukin expression vectors with a DNA immunogen has been demonstrated to improve cellular and humoral immune responses (Chow et al., 1997
; Geissler et al., 1997
; Xiang & Ertl, 1995
). The co-administration of vectors expressing interleukins and flagellin fusions delivered by attenuated Salmonella may improve the stimulation of a specific systemic and mucosal immune response against flagellin and heterologous antigen.
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ACKNOWLEDGEMENTS |
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Received 9 May 2000;
revised 30 October 2000;
accepted 10 January 2001.