INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

Live attenuated vectors are one of the most efficient delivery systems for stimulation of the mucosa-associated immune system (for a review, see reference 10). They have therefore been extensively used to express foreign antigens and epitopes selected from pathogens against which the induction of a local immune response is required for protection (for a review, see reference 23). Usually, foreign epitopes are inserted within a carrier protein that is expressed in the live vector.

Shigella flexneri live attenuated strains have been developed as candidates for vaccines against shigellosis, an invasive disease of the human colon (22). The capacity of such strains to act as mucosal vectors has been recently reported (16). Local and systemic antibody responses to fimbriae and CS3 fibrillae of enterotoxigenic Escherichia coli were generated in guinea pigs or mice following immunization with these antigens expressed in CVD1203, an S. flexneri 2a live attenuated strain that confers protection against Shigella keratoconjunctivitis in the guinea pig model (15).

The purpose of the present study was to investigate whether S. flexneri vaccine strains could be used as immunization vectors to express heterologous epitopes of eukaryotic origin and, in turn, elicit local and systemic antibody responses to foreign sequences. The IpaC antigen, previously reported as a potential carrier protein (1), was selected for the insertion of the neutralizing C3 epitope of the VP1 protein of poliovirus (26). This 11-residue-long sequence has been previously used as a reporter epitope (5, 25). As immunogenicity of a B-cell epitope depends on its flanking sequences within the hybrid protein (25), the C3 epitope was inserted into eight different sites within the IpaC coding sequence. SC602, an S. flexneri 2a vaccine strain attenuated both in its capacity to move intra- and intercellularly and in its survival in tissues (1), was used as vector. This strain is safe and protective in the macaque monkey model (8) as well as in human volunteers (9). The IpaC-C3 hybrid proteins were expressed from recombinant plasmids within SC602 to retain the functionality of the wild-type (wt) IpaC, thus maintaining the invasiveness of the live attenuated vector and ensuring efficient stimulation of local immunity. Immunogenicity of the IpaC-C3 proteins expressed in SC602 was assessed following an immunization protocol that allows the induction of local and systemic anti-IpaC antibody responses in mice (1).

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Bacterial strains and growth media. E. coli MC1061, GM48, and ZK501 (21) were used for construction of plasmids and for demethylation of StuI and BclI restriction sites, respectively. The S. flexneri 2a derivative SC602 [icsA (iuc iut)] (1) was used for expression of the hybrid proteins, and the S. flexneri 5 derivative SF635 [(ipaB ipaC ipaD ipaA)] (19) was used to recover high amounts of hybrid proteins in the culture supernatant. Bacteria were grown in Trypticase casein soy broth supplemented with ampicillin (100 µg/ml) when necessary.

Construction of hybrid proteins. Synthetic oligonucleotides corresponding to the C3 coding sequence were inserted within each of the selected ipaC restriction sites. Plasmid pC1, a pUC derivative containing the ipaC gene (14), was used for C3 insertion within the StuI and AflII sites. Plasmid pC2, generated by removing the EcoRI site present in the pC1 polylinker sequence, was used for C3 insertion within EcoRI site as well as the BclI, PstI, SacI, and NsiI sites. Finally, plasmid pC3, constructed by removing the BglII site located upstream from the IpaC coding sequence in pC2, was used for C3 insertion within the BglII site. The resulting plasmids were designated pC57, pC83, pC123, pC150, pC187, pC267, pC300, and pC351, with the number indicating the position of the insert within the IpaC sequence (Fig. 1A). For the corresponding hybrid proteins, "pC" was replaced by "IpaC". Transformations were performed as previously described (21).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Construction of IpaC-C3 hybrid proteins. (A) C3 insertion sites (the restriction site used for cloning and the resulting position of C3 within the IpaC sequence are indicated by arrows), size of the insert, and modification of IpaC sequence resulting from genetic manipulation. (B) Amino acid residues and corresponding DNA sequence of the C3 epitope. (C) Amino acid residues and corresponding DNA sequences of IpaC-C3 hybrid proteins for each insertion site, indicated as boldface characters inside parentheses for C3, boldface characters outside parentheses for IpaC, and lightface characters for additional sequences resulting from cloning. IpaC residue numbers are indicated.

Protein analysis. Proteins present in whole-cell extracts and culture supernatants of SC602 derivatives were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Those present in culture supernatants of SF635 derivatives were analyzed by SDS-PAGE and enzyme-linked immunosorbent assay (ELISA). Culture dilutions were performed considering that for an optical density at 600 nm (OD₆₀₀) of 1, the bacterial concentrations were 5 × 10⁸ and 3 × 10⁸ CFU/ml for S. flexneri and E. coli, respectively. Exponential-phase (OD₆₀₀ = 1.5) or stationary-phase (OD₆₀₀ = 7) bacterial cultures were harvested, and whole-cell bacterial extracts were obtained after solubilization of bacterial pellets with Laemmli buffer (11). Supernatants from exponential-growth cultures of SC602 derivatives were recovered, filtered, and concentrated 100-fold in Laemmli buffer as already described (2). Supernatants of SF635 derivatives were prepared similarly and were used without being concentrated for ELISA and diluted twice in Laemmli buffer for SDS-PAGE analysis.

Immunization of mice and analysis of the antibody responses. Seven-week-old BALB/c female mice (Janvier, Le Genest, France) were immunized intravenously (i.v.) with formaldehyde-killed SC602 bacteria (at stationary phase) expressing each of the IpaC-C3 proteins (10⁸ CFU/mouse, three times at 15-day intervals). Intranasal immunizations with live bacteria (5 × 10⁶ CFU/mouse, three injections at 15-day intervals) were performed beginning 15 days after the last i.v. immunization. Wild-type SC602 and SC602(pC1) were used as controls. Ten mice were used per group, and experiments were repeated three times. Immunizations with the IpaC300 protein, purified by using a protocol previously described for the purification of IpaC (7), were performed as follows. Nineteen 7-week-old BALB/c female mice were immunized subcutaneously with 25 µg of IpaC300 or IpaC (used as a control) in the presence of incomplete Freund's adjuvant, followed by two i.v. immunizations without adjuvant at 15-day intervals.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Construction of the hybrid proteins. Hybrid ipaC-C3 genes were constructed by inserting synthetic oligonucleotides corresponding to the C3 coding sequence within each of the following ipaC restriction sites: BclI, BglII, PstI, StuI, AflII, SacI, NsiI, and EcoRI (Fig. 1A). All of the C3-encoding oligonucleotides, except those used for cloning within the StuI and AflII sites, were designed with a HindIII restriction site located at the 3' end (Fig. 1B). Therefore, the size of the inserted segment slightly exceeds the size of C3 coding sequence as a result of genetic manipulations and introduction of an additional leucine codon at the end of the C3 coding sequence. This codon is present in all constructs except that in which the C3 epitope was inserted within the StuI site (Fig. 1C). All of the hybrid proteins contain the entire sequence of IpaC except for IpaC123, which lacks residues 124 to 142, and IpaC300, which lacks residue 300 (Fig. 1A).

Production and secretion of IpaC-C3 proteins in SC602. The production and stability of the IpaC-C3 proteins expressed within the vaccine candidate strain SC602 during exponential growth, in stationary phase, and after formaldehyde killing were analyzed by SDS-PAGE (11) and immunoblotting of whole bacterial extracts as described in Materials and Methods. All hybrid proteins were produced at a level comparable to that of wt IpaC expressed in SC602(pC1) (Fig. 2A). Using the anti-IpaC MAb, we detected two bands that correspond to the hybrid protein (upper band) and to the wt IpaC (lower band). Since the molecular weights of IpaC123 and IpaC are almost identical, the two bands were not separated in SC602(pC123) whole bacterial extract. However, production of this recombinant protein was confirmed by using the anti-C3 serum (Fig. 2B). Comparison of the relative intensities of the signals obtained for each protein with the anti-IpaC and anti-C3 sera indicated that recognition of the C3 epitope within the IpaC-C3 hybrid proteins depends on the insertion site (Fig. 2B). Overproduction of the hybrid proteins from the recombinant plasmids led to their accumulation within bacteria harvested in stationary phase (Fig. 2C). Similarly, the hybrid proteins remained stable after formaldehyde treatment of the bacteria (data not shown).

View larger version (60K): [in this window] [in a new window]   FIG. 2.   Production of IpaC-C3 hybrid proteins in SC602. (A, B, and D) Detection of IpaC-C3 hybrid proteins in whole-cell extracts with the anti-IpaC K24 MAb (A) or anti-C3 polyclonal serum (B) and in culture supernatants with anti-C3 serum (D) by immunoblot analysis of exponentially grown cultures. (C) SDS-PAGE and Coomassie blue staining of whole-cell extracts of stationary-phase cultures after formaldehyde treatment. The arrowhead indicates the position of the hybrid proteins.

Antigenicity of the IpaC-C3 hybrid proteins. Western blot experiments performed with anti-C3 antibodies suggested that the hybrid proteins exhibited variable antigenicity with respect to the C3 epitope (Fig. 2). Therefore, we studied the accessibility of this epitope in comparison of two linear epitopes of IpaC, J22 and K24, which are located between residues 25 to 33 and 300 to 349, respectively. The accessibility of C3, J22, and K24 epitopes was assessed by ELISA as described in Materials and Methods, using each of the hybrid proteins for coating. To easily obtain large amounts of the hybrid proteins in the culture supernatant, we expressed each of them in S. flexneri SF635 (19). In this strain, in contrast to SC602, secretion of IpaC and consequently IpaC-C3 proteins is not controlled by IpaB and IpaD, which regulate the Mxi-Spa secretion machinery. Therefore, each hybrid protein, even IpaC83, which is not efficiently secreted in SC602, is found in the culture supernatant.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Antigenicity of C3 within IpaC-C3 hybrid proteins. Accessibility of the C3 epitope is expressed as arbitrary units corresponding to the ratio (anti-C3 antibody titer/anti-K24 antibody titer) × 100.

Identification of permissive insertion sites within IpaC that allow induction of anti-C3 humoral responses. To identify insertion sites within IpaC that allow induction of a systemic anti-C3 antibody response, mice were immunized i.v. with killed (formaldehyde-treated) SC602 expressing each of the IpaC-C3 as described in Materials and Methods. SC602 and SC602(pC2) were used as controls. Antibody responses were assessed by ELISA (see Materials and Methods). The results are shown in Fig. 4. As expected, anti-IpaC antibodies were induced following immunization with each of the recombinant strains. The titer obtained following immunization with SC602(pC2) was higher than that obtained following immunization with SC602 (Fig. 4A). This result is consistent with the higher production of IpaC in SC602(pC2), in which IpaC is expressed from both the virulence plasmid and a recombinant plasmid (Fig. 2C). Five of the eight strains induced anti-IpaC antibody titers similar to that induced upon immunization with SC602(pC2). In contrast, anti-IpaC antibody titers raised upon immunization with SC602(pC83), SC602(pC150), and SC602(pC351) were similar to that of wild-type SC602. Differences in the secretion efficiency or in the amount of proteins could not account for the differences observed in the titers, as priming was achieved with killed bacteria, and all strains produced similar levels of hybrid proteins (Fig. 2A). Differences in the ability to induce anti-IpaC antibodies have also been observed when another foreign epitope was inserted within two different sites of IpaC (3). Since no major conformational modifications of IpaC could be detected following C3 insertion (2), these results suggest that insertions within a protein carrier may influence its own immunogenicity without significantly affecting its overall conformation.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Anti-C3 and anti-IpaC serum IgG antibody titers in mice immunized with SC602 producing IpaC-C3 hybrid proteins. (A) Mean antibody titers in serum samples are shown for each group of mice immunized with each of the IpaC-C3 hybrid proteins expressed in SC602. SC602(pC2) and SC602 were used as controls. SD is indicated. The results are representative of three experiments, each using 10 mice per group. (B) Individual anti-C3 serum IgG antibody titers in SC602(pC267)- and SC602(pC300)-immunized mice.