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The use of attenuated Salmonella strains as live vector vaccines to deliver foreign antigens to the mammalian immune system is an exciting area of vaccinology that has great potential. Previously, we utilized attenuated Salmonella typhi expressing fragment C of tetanus toxin and the S1 subunit of pertussis toxin to stimulate, respectively, serum antitoxin that neutralizes tetanus and pertussis toxins following mucosal (intranasal) immunization of mice (3, 7). The success of that approach led us to attempt the same with diphtheria toxin, the long-term goal being an S. typhi-based mucosally administered vaccine against diphtheria, pertussis, and tetanus.

Diphtheria toxin (DT), a 535-amino-acid protein encoded by tox of Corynebacterium diphtheriae, is secreted as a single molecule of 58,350 Da encompassing two functional subunits, subunit A, the catalytic domain responsible for the ADP-ribosylation activity of the toxin in eukaryotic cells, and subunit B, the trans-membrane and receptor-binding domains. Human antibodies raised by immunization with diphtheria toxoid react with both subunits A and B (4, 19), and monoclonal antibodies to both subunits can neutralize DT (5, 20, 23, 24). Nevertheless, most neutralizing antitoxin is anti-B subunit and inhibits binding of the toxin to its receptor. A neutralizing epitope has been described corresponding to a cysteine loop (residues 186 to 201) located between the A and B subunits (1).

Because of its potent toxicity, either a stable nontoxic mutant protein (i.e., a cross-reacting molecule, or CRM) or noncatalytic fragments would have to be expressed in S. typhi. The most extensively studied nontoxic mutant DT, CRM₁₉₇, which carries a glycine to glutamic acid substitution at residue 52 within the catalytic domain (8), can induce neutralizing antitoxin (11). We investigated attenuated S. typhi vaccine strain CVD 908-htrA (15, 22) as a live vector to deliver diphtheria antigens and induce protective antitoxin in animal models.

Initial attempts to express CRM₁₉₇ holotoxin in CVD 908-htrA. Toward the goal of expressing relevant diphtheria toxin epitopes within CVD 908-htrA, we undertook two parallel approaches involving both expression of the full-length nontoxic mutant holotoxin CRM₁₉₇ and expression of domains or fragments of CRM₁₉₇ in an attempt to increase the levels of synthesis of neutralizing epitopes. With p197 as the template, we used PCR to synthesize three BglII-NheI cassettes encoding full-length unmodified CRM₁₉₇ (creating pNO1), CRM₁₉₇ into which an optimized ribosome binding site and start codon were engineered (creating pNO2), and CRM₁₉₇ from which the signal sequence was removed (creating pNO3). These constructions are represented graphically in Fig. 1; all primers are summarized in Table 1, and plasmids are summarized in Table 2.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Illustration of DT derivatives used in this study. The engineering of fragments encoding CRM197 or individual domains of this protein were carried out by PCR and Vent DNA polymerase (New England BioLabs, Inc., Beverly, Mass.) with the plasmid template pB197 which carries the promoter, signal sequence, and full-length mutant CRM197 structural gene. Primers used in the construction of the plasmids employed in this study are described in Table 1 and were designed by using the published sequence of the diphtheria tox gene encoded by corynebacteriophage  (10) (GenBank accession no. K01722). PCR products synthesized with Vent polymerase were then treated with Taq DNA polymerase to generate the 3' deoxyadenosine necessary for direct cloning into the plasmid pGEM-T (Promega Corp., Madison, Wis.); recombinant plasmids were recovered by transformation into MAX Efficiency E. coli DH5 frozen competent cells (Gibco BRL, Gaithersburg, Md.). Restriction endonuclease sites incorporated into the primers were then used to subclone fragments into plasmids which were introduced into attenuated S. typhi live vector CVD 908-htrA, using electroporation as previously described (9). Detailed descriptions of the plasmids used in this work are listed in Table 2, and CRM197 derivatives generated are graphically represented. Most PCR products were subcloned into pTETnir15, either replacing the gene encoding the nontoxic fragment C of tetanus toxin or resulting in synthetic genes encoding protein fusions of fragment C and CRM197 or various subdomains. In an attempt to enhance the solubility of potentially relevant neutralizing epitopes of diphtheria toxin, a fragment encoding amino acids 186 to 201 of diphtheria toxin (1) was synthesized and fused in-frame as two copies to the carboxyl terminus of fragment C to create a repitope (14). In addition, we attempted to achieve secretion of CRM197 from CVD 908-htrA by inserting the open reading frame encoding CRM197 in-frame into the unique NsiI site of a truncated version of hlyA encoding Hemolysin A within the plasmid pMOhly (13).

View larger version (71K): [in this window] [in a new window]   FIG. 2.   Western immunoblot analysis of soluble (s) and insoluble (i) fractions from CVD 908-htrA-expressing CRM197 or its derivatives FC-DTA197 and eDTB197. Membranes were probed with anti-DT antibodies. Lanes D, DT (5 pg); lane 1, pNO1; lane 2, pNO2; lane 3, pNO3; lane 4, pNO7; lane 5, pNO8. E. coli DH5 carrying recombinant plasmids was grown with Luria broth base (LB) medium (Gibco BRL) supplemented with 50 µg of carbenicillin (Sigma, St. Louis, Mo.)/ml. For expression studies, S. typhi CVD 908-htrA was streaked from frozen stocks onto LB agar supplemented with 0.0001% (wt/vol) 2,3-dihydroxybenzoic acid (DHB; Sigma) and 50 µg of carbenicillin/ml where appropriate. Isolated colonies were then inoculated into LB broth containing DHB and carbenicillin and incubated overnight at 37°C, 250 rpm. For expression under aerobic conditions, late-logarithmic or stationary-phase cultures were diluted 1:100 into fresh LB broth and again incubated overnight at 37°C, 250 rpm; for anaerobic induction experiments, Oxyrase solution (Oxyrase, Inc., Mansfield, Ohio) was added to identical LB broth cultures and incubated static at 37°C overnight. Selected CVD 908-htrA strains expressing significant levels of CRM197-derived proteins were grown as previously described (7) for immunization of mice. The 150-ml cultures were grown to an optical density at 600 nm, ~1.0 were centrifuged, and bacterial pellets were resuspended in 3.5 ml of ice-cold sonication buffer (phosphate-buffered saline containing 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 0.1% Tween 20, and 20 mM -mercaptoethanol) for solubility studies. Bacterial suspensions were disrupted by sonication for 5 cycles of 20 s on ice by using a model 550 sonic dismembrator (Fisher Scientific, Pittsburgh, Pa.) with a microtip and a power level of 5. Sonicates were centrifuged at 15,000 rpm for 30 min at 4°C, and supernatants representing the soluble fraction were removed; cell pellets were reconstituted with 3.5 ml of sonication buffer to represent the insoluble fraction. Proteins were then heat denatured after equal volumes of sample and lysis buffer were mixed, and proteins from 5 µl of each denatured sample were then separated by SDS-PAGE with 10% polyacrylamide gels. Separated proteins were detected either by staining with BLUPRINT Fast-PAGE Stain (Gibco BRL) or by being transferred to Immun-Lite blotting membrane (Bio-Rad Laboratories, Hercules, Calif.) for Western immunoblot analysis. CRM197-derived proteins were detected by using polyclonal goat anti-DT serum (Biogenesis, Sandown, N.H.), and fragment C fusions were confirmed by using monoclonal mouse anti-fragment C antibodies (Boehringer Mannheim, Indianapolis, Ind.). Membranes were then incubated with horseradish peroxidase-conjugated rabbit anti-goat (Sigma) or peroxidase-conjugated goat anti-mouse IgG (Gibco BRL) as appropriate. Immunoblots were developed by chemiluminescence with an ECL Western blotting kit (Amersham Life Science Inc., Arlington Heights, Ill.), and signals were detected with X-OMAT XAR-5 film (Eastman Kodak Company, Rochester, N.Y.).

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Expression of domains or fragments of CRM₁₉₇ in CVD 908-htrA. Since the solubility and immunogenicity of full-length CRM₁₉₇ expressed within CVD 908-htrA appeared problematic, we expressed various domains of the holotoxin in an attempt to enhance the expression of soluble antigen. Initial attempts to express fragment A of CRM₁₉₇ by simply replacing the BglII-NheI gene cassette encoding fragment C in pTETnir15 to create pNO6 were unsuccessful, probably due to proteolytic degradation (data not shown). We constructed another cassette encoding mature DTA₁₉₇ without the signal sequence as a HindIII-BamHI cassette, which was inserted into the expression vector pOG214 (9) to create pNO7, which now carries a synthetic gene encoding the protein fusion of mature DTA₁₉₇ fused to the carboxyl terminus of fragment C and separated by a 4-amino-acid hinge region (Fig. 1). This approach was previously used to rescue expression of the receptor binding domain of CRM₁₉₇ (9). In a related approach, we constructed pNO8 to express a truncated holotoxin in which the amino-terminal 53 amino acids including the catalytic site of the holotoxin were deleted; this construct therefore encodes an extended version of the DTB subunit, which we refer to here as eDTB₁₉₇. Both re-engineered genes were expressed at high levels in CVD 908-htrA when grown anaerobically, and the fusion proteins were readily detected both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels stained for total protein (data not shown) and with anti-DT antibodies by Western immunoblot analysis (Fig. 2, lanes 4 and 5). However, the solubility of these protein fusions did not improve. Serum anti-DT was not detected in mice immunized intranasally with CVD 908-htrA(pNO7) or CVD 908-htrA(pNO8), and neutralizing antitoxin was not detected in guinea pigs immunized with extracts from these strains.

View larger version (68K): [in this window] [in a new window]   FIG. 3.   Western immunoblot analysis of soluble (s) and insoluble (i) fractions of CVD 908-htrA expressing fragment C fused in-frame to a repitope of DT residues 186 to 201 or to tDT197. Membranes were probed with anti-DT or anti-FC antibodies. Lanes: T, pTETnir15; 1, pNO12; 2, pNO11; 3, pNO10; D, DT (5 pg).

Expression of CRM₁₉₇ by using the HlyA expression system. Since inclusion of the native signal sequence failed to drive expression of a soluble product within CVD 908-htrA(pNO1) and CVD 908-htrA(pNO2), we used an alternate secretion mechanism derived from E. coli, in which heterologous antigens are inserted in-frame into a truncated version of the hemolysin A protein and potentially secreted by the hemolysin secretion apparatus (13). For this purpose, the gene encoding mature CRM₁₉₇ holotoxin was re-engineered to include the appropriate restriction sites and inserted in-frame as a PstI-NsiI cassette into the unique NsiI site within the truncated hlyA gene of pMOhly, creating pNO13 (Fig. 1). In this case, the majority of the HlyA-CRM₁₉₇ fusion product was soluble, although no product was detected in the medium (Fig. 4, lane s versus lanes m and i). However, the level of expression was low compared to that induced by pNO3. Despite this low level of expression, we examined the immunogenicity of this fusion protein in mice immunized intranasally with CVD 908-htrA(pNO13). No serum antibody response against DT was detected.

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Western immunoblot analysis of CVD 908-htrA(pNO13). Membranes were probed with anti-DT antibodies. Lanes: m, medium; s, soluble fraction; i, insoluble fraction; w, whole cell; D, DT (5 pg).