Print version ISSN 0074-0276
Mem. Inst. Oswaldo Cruz vol.103 no.6 Rio de Janeiro Sept. 2008
LM MassisI, +; CJM BragaI; ME Sbrogio-AlmeidaII; C LauandI; SMC NewtonIII; PE KlebbaIII; LCS FerreiraI
IDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-000 São Paulo, SP, Brasil
IIDivisão de Desenvolvimento Tecnológico e Produção, Instituto Butantan, São Paulo, SP, Brasil
IIIDepartment of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.
Key words: fagellin - antibody response - attenuated S. Typhimurium strains
Attenuated Salmonella enterica strains have been studied intensively as live carriers of heterologous vaccine antigens delivered by mucosal or parenteral routes (Brey et al. 1991, Garmory et al. 2002). Orally delivered, attenuated Salmonella strains induce both systemic and secretory immune responses against the carrier strain as well as the heterologous passenger antigen (Garmory et al. 2002). Additionally, peptides, particularly those encoding B-cell epitopes, may be expressed by attenuated Salmonella strains as hybrid proteins following genetic fusion to bacterial proteins. This approach may improve both the stability and the immunogenicity of target antigens delivered orally in live bivalent vaccines (Dougan et al. 1987, Chatfield et al. 1992).
The fusion of heterologous antigens to Salmonella flagellin, the structural subunit of bacterial flagella, has piqued considerable interest as an approach to enhancing the immunogenicity of vaccine peptides (Newton et al. 1989, Stocker 1990, Newton et al. 1991, Stocker & Newton 1994). As originally proposed, the heterologous peptide is fused in-frame to the central hypervariable domain of Salmonella FliCd flagellin, which is derived from Salmonella Müenchen and expressed by an attenuated Salmonella Dublin strain. The chimeric flagellins are exported to the bacterial surface where the subunits assemble into the flagellar shaft without a significant impact on bacterial motility and host tissue colonization (Newton et al. 1989, Stocker & Newton 1994). Nonetheless, our previous results showed that the genetic fusion of heterologous peptides to flagellins may not enhance antigen-specific antibody responses in mice orally immunized (p.o.) with recombinant S. Dublin vaccine strains (De Almeida et al. 1999, Sbrogio-Almeida & Ferreira 2001). Interestingly, the genetic background of both the mouse lineage and the Salmonella strain affected the immunogenicity of flagellins delivered orally using recombinant vaccine strains (Sbrogio-Almeida 2004). Indeed, recent evidence indicates that Salmonella flagellin administered via the oral route may trigger immunological hyporesponsive responses in healthy mice, although the precise mechanism underlying these responses remains unknown (Sanders et al. 2006).
In contrast to S. Dublin strains, wild-type S. Typhimurium strains express two flagellar phases: phase 1 (FliCi) and phase 2 (FljB), which are alternately produced by a single cell but may be found concurrently in the same bacterial population (Ikeda et al. 2001, Yamamoto & Kutsukake 2006). So far, the oral immunogenicity of the two S. Typhimurium flagellins, as well as their role as potential heterologous peptide carriers, has not been investigated. To explore these issues, the present study analyzed the antibody-inducing properties of flagellins expressed by the attenuated S. Typhimurium SL3261 strain.
The bacterial strains and plasmids used in this study are described in Table I. Recombinant plasmids were introduced by electroporation into the non-flagellated S. Typhimurium strain LDV321. The plasmid pFF400 is a pUC18 derivative carrying the fliC gene of SL3261. pFF408 was constructed by replacing a 48 bp region between two natural EcoRV sites with a polylinker containing unique XhoI and BglII sites at the central region of FliCi-encoding gene (Newton et al. 1989). pFF302 is a pFF408 derivative that carries a hybrid fliCi gene encoding a 15-amino acid peptide (VDPVIDLLQADGNAL). This peptide defines B and T-cell epitopes of the structural subunit (CfaB) of the CFA/I fimbriae from enterotoxigenic Escherichia coli (ETEC) (Luna et al. 1997) at the FliCi central hypervariable domain. Similarly, pFF411 delivers a recombinant fliCi gene encoding the 15 amino acid-long CTP3 peptide (VEVPGSQHIDSQKKA) derived from the B subunit of the cholera toxin (Newton et al. 1989) at the same flagellin domain.
Flagellin-deficient S. Typhimurium strains have been generated following chromosomal deletions of each native flagellin gene (fliCi and fljB) by allelic replacement (Datsenko & Wanner 2000). Primers were designed based on the flagellin sequences of the S. Typhimurium LT2 strain (McClelland et al. 2001). Deletion of fljB also included the fljA gene, which encodes the fliC gene transcriptional repressor. The amplified fragments were introduced by electroporation into the attenuated SL3261 strain transformed with the arabinose-inducible red genes-encoding plasmid (pKD46), previously propagated in the restriction minus (r-m+) metilation plus S. Typhimurium TT22971 strain. The phage lambda recombinase allows high-frequency recombination between linear DNA fragments generated by polymerase chain reaction (PCR) and homologous regions in the bacterial chromosome (Datsenko & Wanner 2000). To avoid undesirable mutations and to obtain the double-deletion mutant (ΔfliC, ΔfljBA) the strains were submitted to transductional crosses using a high frequency-transducing P22 phage (HT105 int) as previously described (Schmieger 1971). The strains were also transformed with pCP20, encoding resistance to ampicillin and chloramphenicol, to remove the resistance cassettes inserted in the target chromosomal sites. As previously reported, this plasmid undergoes temperature-sensitive replication and induction of flp (the gene encoding the Saccharomyces cerevisiae Flp recombinase) synthesis after a temperature shift (Cherepanov & Wackernagel 1995). The genomic deletions of the resulting S. Typhimurium LDV322 (ΔfljBA), LDV323 (ΔfliC) and LDV321 (ΔfliC, ΔfljBA) strains were confirmed by PCR and immunoblot assays. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed according to standard procedures (Sbrogio-Almeida & Ferreira 2001). Motility was assessed by applying the bacterial strains to the center of plates containing 0.35% agar, followed by incubation at 37°C for 32 h. Motility agar plates were prepared according to previously established methods (Ikeda et al. 2001).
Flagellins expressed by S. Typhimurium LDV322 or LDV323 were harvested from cultures incubated overnight under mild aeration (80 rpm) at 37°C. Cells were collected by centrifugation, suspended in phosphate-buffered saline (pH 7.4) and homogenized for 2 min using a bench mixer. The cells were maintained on ice and the above procedure was repeated three times. The mixture was then centrifuged at 10,000 g for 10 min to remove the bacteria and flagella were collected from the supernatant following acetone precipitation. Contaminating lipopolysaccharide (LPS) was removed using a gel-detoxi column (Pierce). Recombinant His-tagged FliCi flagellin was produced in E. coli transformed with pET28a carrying the cloned fliCi gene. Protein concentration was measured using the BCA quantification method (Pierce).
Isogenic, pathogen-free 8-12 week old female BALB/c mice were supplied by the Isogenic Mouse Breeding Facility of the Departamento de Parasitologia do Instituto de Ciências Biomédicas da Universidade de São Paulo and all procedures were approved by the ethics committee on use of laboratory animals. Immunizations were carried out using viable bacterial strains harvested during exponential growth (optical density at 600 nm of 0.8). Mice were p.o. with 0.5 ml aliquots of approximately 1010 CFU using a stainless steel, round-tip gavage cannule on days 0, 21 and 35. The same immunization schedule was followed for the intraperitoneally (i.p.) immunizations, but the bacterial loads were reduced to 107 CFU per dose. Serum and fecal wash samples were collected individually, but samples belonging to the same immunization groups (with 5-10 individuals) were pooled for antibody titer determination (Sbrogio-Almeida & Ferreira 2001).
Microtiter plates (Nalge Nunc) were coated (0.1 µg/well) with purified FliCi and FljB or the CfaB (Luna et al. 1997) and CTP3 oligopeptides (De Almeida et al. 1999) in 0.05 M sodium bicarbonate buffer (pH 9.6) overnight at RT. Experimental procedures for detecting specific serum IgG and fecal IgA responses have been described previously (Sbrogio-Almeida et al. 2004).
Isogenic flagellin-deficient knockout mutants of the attenuated S. Typhimurium SL3261 strain, affected in either fliCi, fljB or both genes, were selected after homologous recombination mediated by the bacteriophage lambda recombinase encoded by the red genes (Fig. 1). In vitro tests carried out with the flagellin-deficient derivatives of the S. Typhimurium SL3261 strain showed that deletion of either fliCi or fljB did not impair the motility of the respective strains. Nonetheless, as expected, deletion of both flagellin-encoding genes rendered the LDV321 strain non-motile (Fig. 1).
Initial attempts to measure the flagellin-specific antibody responses in vaccinated mice indicated that mice immunized with the S. Typhimurium SL3261 or LDV321 strains mounted similar systemic and secreted anti-flagellin antibody responses following oral administration (Fig. 2). As demonstrated in Fig. 3, removal of LPS from flagellin preparations drastically reduced the reactivity of sera in mice immunized with the Salmonella strains. Indeed, the recombinant FliCi flagellin, produced in E. coli, reacted weakly with the antibodies generated in mice p.o. with the SL3261 strain (Fig. 3). Additionally, western blots carried out with Salmonella flagellin preparations and sera from mice vaccinated with the S. Typhimurium SL3261 strain showed that, besides the contaminant LPS present in flagellin preparations, a minor protein with an estimated molecular mass of 35 kDa and not detected in Coomassie blue-stained gels reacted with the tested serum sample (Fig. 3).
The following Enzyme Linked Immuno Sorbent Assay (ELISA) measurements were thus carried out using LPS-free Salmonella flagellin preparations. The results obtained after the p.o. trials with the S. Typhimurium SL3261, LDV322 or LDV323 strains indicated that no significant flagellin-specific serum IgG responses were induced in mice immunized with the flagellated S. Typhimurium strains (Table II). Similar conclusions were drawn following measurement of flagellin-specific IgA in fecal extracts of mice p.o. with flagellated S. Typhimurium vaccine strains (data not shown). In contrast, mice immunized via the i.p. route with flagellated S. Typhimurium strains developed high FliCi or FljB-specific serum IgG responses following vaccination with the LDV322 or LDV323 strain, respectively (Table II). Collectively, these results indicate that mice p.o. with flagellated S. Typhi-murium strains did not develop significant systemic or secreted flagellin-specific antibody responses.
To determine if the lack of antibody responses to S. Typhimurium flagellins observed in mice p.o. immunized with the attenuated LDV3212 or LDV325 strain would affect antibody responses to heterologous epitopes genetically fused at the central hypervariable region of FliCi, we measured the systemic (serum IgG) and secreted (fecal IgA) responses to peptides derived from the ETEC CFA/I fimbriae structural subunit and the B subunit of cholera toxin. Although the presence of the flagellin inserts could be confirmed by either DNA sequencing or western blot with anti-flagellin serum (Fig. 1B and unpublished observations), BALB/c mice p.o. with the recombinant S. Typhimurium strains did not develop significant serum or fecal antibody responses to the he-terologous epitopes (data not shown).
Using the isogenic, flagellin-deficient S. Typhimurium strains, it was possible to show that the previously detected flagellin-specific serum antibody responses elicited in mice p.o. with flagellated strains were due to the presence of contaminating LPS, as well as an outer membrane protein in flagellin preparations used as solid-phase bound antigen. Thus, the generation of flagellin preparations with higher purity and the use of a non-flagellated strain helped us to demonstrate that mice p.o. with flagellated S. Typhimurium strains failed to mount significant systemic and secreted flagellin-specific antibody responses, similar to results previously reported for S. Dublin strains (Sbrogio & Almeida 2001, Sbrogio et al. 2004, Sanders et al. 2006).
Since mice parenterally immunized with flagellated S. Typhimurium strains developed strong antibody responses to flagellins, the observed flagellin-hypor-esponsive state might be restricted to the murine mucosal immune system. Recent findings confirmed that bacterial flagellins do not elicit inflammatory responses in the normal gut epithelium, a behavior explained by the lack of functional TLR5 expression at the apical surface of enterocytes (Gewirtz et al. 2001), the activation of apoptotic responses by flagellin-exposed antigen-presenting cells (Ren et al. 2006) and/or the activation of suppressive CD4+ CD25+ T regulatory cells (Crellin et al. 2005). Although our findings indicate that the expression of chimeric flagellins by recombinant attenua-ted S. Typhimurium strains has an inherently reduced immunogenicity as an oral bivalent vaccine approach, stimulation of specific immune responses triggered by purified flagellins delivered via the nasal or parenteral routes (Honko et al. 2006) represents more promising alternative for the use of Salmonella flagellins in vaccine development.
To Dr. Melissa A S Lasaro (University of Pennsylvania) and Dr. Karina R Bortoluci (Universidade de São Paulo), for helpful comments, and Camila F P Calderon, Carlos A da Silva and Aline F Teixeira, for invaluable technical assistance.
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Received 15 April 2008
Accepted 22 August 2008
Financial support: CNPq, FAPESP, NIH grant GM53836, NSF grant MCB0417694, FAPESP, CAPES