Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA

Abstract

Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

FIG. 1.

Genetic maps of isogenic expression plasmids encoding ClyA and ClyA fusion proteins; a list of the plasmids used in this work is shown in Table 2. Restriction sites shown in bold represent unique sites in the expression plasmids. Abbreviations: P_ompC, modified osmotically controlled ompC promoter from E. coli; clyA, gene encoding ClyA from Salmonella serovar Typhi; gfpuv, gene encoding prokaryotic codon-optimized GFPuv; d4, gene encoding D4 of anthrax toxin PA; clyA::d4, gene encoding D4 of PA fused to the carboxyl terminus of ClyA; tetA, gene encoding the tetracycline efflux protein from pBR322; aph, gene encoding the aminoglycoside 3′-phosphotransferase conferring resistance to kanamycin; oriE1, origin of replication from pBR322 providing an expected expression plasmid copy number of ∼60 per chromosomal equivalent; ori15A, origin of replication from p15A providing an expected copy number of ∼15 per chromosomal equivalent; T1, transcriptional terminator from the rrnB rRNA operon of E. coli; hok-sok, postsegregational killing locus from the multiple antibiotic resistance R plasmid pR1; and parA, gene encoding the active partitioning system from pR1.

FIG. 2.

Western immunoblot analysis of bacterial cell fractions from either CVD 908-htrA (lanes 1 to 3) or CVD 908-htrA(pSEC84gfpuv) (lanes 4 to 8). Cell fractions analyzed are shown above the lanes; lane 9 contains 50 ng of GFPuv. Numbers at left indicate kilodaltons. Membranes with identical samples were probed with antibodies specific for GFPuv (A) or E. coli GroEL (B). Immunoblots were scanned as high-resolution TIFF files, and image contrast was improved by use of Adobe Photoshop 6.0.

FIG. 3.

(A) Coomassie brilliant blue-stained SDS-polyacrylamide gel of whole-cell lysates from either CVD 908-htrA (lane 1), CVD 908-htrA(pSEC91D4) (lane 2), or CVD 908-htrA(pSEC91D4-c) (lane 3); lane 4 contains 0.3 μg of purified PA63. (B) Western immunoblot analysis of whole-cell lysates from either CVD 908-htrA (lane 1), CVD 908-htrA(pSEC91D4) (lane 2), or CVD 908-htrA(pSEC91D4-c) (lane 3); lane 4 contains 0.3 μg of purified PA63. Membranes were probed with murine polyclonal antiserum raised against D4 as described in Materials and Methods. The expected molecular mass of D4 expressed within the cytoplasm was 16.5 kDa; the expected molecular mass of D4 expressed as a ClyA-D4 fusion protein was 50.2 kDa. Immunoblots were scanned as high-resolution TIFF files, and image contrast was improved by use of Adobe Photoshop 6.0. (C) Western immunoblot analysis of whole-cell lysates and supernatants from either CVD 908-htrA(pSEC91D4-c) (lanes 1 to 3 and 8) or CVD 908-htrA(pSEC91D4) (lanes 4 to 6 and 9); lane 7 contains 0.5 μg of purified PA63. Supernatants were harvested from CVD 908-htrA(pSEC91D4-c) cultures grown to OD₆₀₀s of 0.54, 0.95, and 1.65 (lanes 1 to 3, respectively) and from CVD 908-htrA(pSEC91D4) cultures grown to OD₆₀₀s of 0.53, 0.92, and 1.47 (lanes 4 to 6, respectively). Immunoblots were analyzed as described for panel B. Lanes M in all panels contain molecular mass markers (in kilodaltons).