Use of mchI encoding immunity to the antimicrobial peptide microcin H47 as a plasmid selection marker in attenuated bacterial live vectors

Abstract

Live attenuated bacterial strains expressing heterologous antigens represent an attractive vaccine development strategy. However, the use of drug resistance genes for the selection of expression plasmids introduced into live vectors poses theoretical health risks. Therefore, we developed a novel approach for plasmid selection based on immunity to the antimicrobial peptide microcin H47 (MccH47). Two expression plasmids encoding the reporter green fluorescent protein (GFPuv) were constructed; selection markers comprised either mchI, conferring immunity to MccH47 (pGEN222I), or bla (encoding beta-lactamase), conferring conventional resistance to ampicillin (pGEN222). GFPuv-specific serum immunoglobulin G (IgG) antibody responses were analyzed in mice immunized intranasally either with Salmonella enterica serovar Typhi CVD 908-htrA or Shigella flexneri 2a CVD 1208S live vector and were boosted parenterally with purified GFPuv. Similar IgG antibody responses were observed for both pGEN222 and pGEN222I when either CVD 1208S or CVD 908-htrA(pGEN222I) was used as the carrier. Interestingly, CVD 908-htrA(pGEN222I) elicited a significantly higher IgG response than CVD 908-htrA(pGEN222). We also compared the priming potential of homologous priming either with CVD 908-htrA(pGEN222I) or CVD 1208S(pGEN222I) to heterologous priming first with CVD 908-htrA(pGEN222I) and then with CVD 1208S(pGEN222I) and vice versa. Immunization with two unrelated live vectors significantly enhanced the IgG responses compared to responses engendered by homologous CVD 908-htrA(pGEN222I) but not to those of CVD 1208S(pGEN222I). MccH47 offers an alternate system for plasmid selection in bacterial live vectors that greatly improves their clinical acceptability. Furthermore, the success of the heterologous priming strategy supports the feasibility of the future development of multivalent live vector-based immunization strategies against multiple human pathogens.

FIG. 1.

Genetic maps of isogenic expression plasmids encoding the prokaryotic codon-optimized GFPuv. Key restriction sites used for constructions are shown. Abbreviations: P_ompC, modified osmotically controlled ompC promoter from E. coli; gfpuv, gene encoding prokaryotic codon-optimized GFPuv; T1, transcriptional terminator from the rrnB rRNA operon of E. coli; par, passive partitioning function from pSC101; ori15A, origin of replication from p15A providing an expected copy number of ∼15 per chromosomal equivalent; bla, β-lactamase gene conferring resistance to ampicillin; mchI, gene coding for the immunity protein MchI conferring resistance to MccH47; hok-sok, postsegregational killing locus from the multiple antibiotic resistance R plasmid pR1; and parM and parR, two loci comprising the parA active partitioning system from pR1.

FIG. 2.

MccH47 sensitivity assays for CVD 908-htrA carrying plasmids pGEN222 (lanes 1), pGENMch (lanes 2), pGENMchK2 (lanes 3), and pGEN222I (lanes 4). (A and B) The cross-streaking method for resistance to MccH47. An overnight culture of RYC1000(pEX4) was swabbed from left to right across 2× LB50 plates and allowed to grow overnight at 37°C to impregnate the medium with MccH47 (A, dotted horizontal rectangle). Excess bacterial growth then was removed from the surface, and remaining bacteria were lysed with chloroform. CVD 908-htrA carrying plasmids expressing GFPuv with or without MchI then were streaked orthogonally to the MccH47 zone (A, vertical green rectangle), and plates were again incubated overnight at 37°C. The presence or absence of zones of clearing near the MccH47 region are represented as dotted circles in the graphic; panel B documents these zones for plasmid-bearing CVD 908-htrA live vectors. (C and D) Patch tests for resistance to MccH47. 2× LB50 plates were seeded with a lawn of CVD 908-htrA carrying plasmids expressing GFPuv with or without MchI (C, green circle). The MccH47-expressing strain RYC1000(pEX4) was spotted in the middle of each plate, and plates were incubated at 37°C overnight. The presence or absence of zones of clearing are represented by dotted circles; panel D documents these zones for plasmid-bearing CVD 908-htrA live vectors.

FIG. 3.

In vitro stability of pGEN222 and pGEN222I plasmids in CVD 908-htrA and CVD 1208S live vectors. Live vector strains were passaged at 37°C from an overnight starter culture (grown without selection) every 24 h for 96 h in antibiotic-free liquid medium. Plasmid stability is reported as the percentage of fluorescing colonies from the total number of CFU plated on nonselective medium.

FIG. 4.

Western immunoblot analysis of whole bacterial lysates of CVD 908-htrA (lane 1), CVD 908-htrA(pGEN222) (lane 2), CVD 908-htrA(pGEN222I) (lane 3), CVD 1208S (lane 4), CVD 1208S(pGEN222) (lane 5), CVD 1208S(pGEN222I) (lane 6), and GFPuv protein (50 ng) (lane 7). Numbers at the left denote relative molecular masses in kilodaltons. The detection of GFPuv was carried out using murine polyclonal primary antibody.