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Published on: March 16, 2012
Use of mchI encoding immunity to the antimicrobial peptide microcin H47 as a plasmid selection marker in attenuated
Chee-Mun Fang1, Jin Yuan Wang, Magaly Chinchilla
1Department of Medicine, Division of Geographic Medicine, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. cfang@medicine.umaryland.edu
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.
Insights
This study introduces a novel plasmid selection method using microcin H47 (MccH47) immunity for live bacterial vaccines, enhancing safety and efficacy. This approach supports developing multivalent vaccines against various pathogens.
Area of Science:
- Microbiology
- Vaccinology
- Immunology
Background:
- Live attenuated bacterial strains are promising vaccine vectors.
- Traditional plasmid selection using drug resistance genes poses health risks.
- A safer alternative for plasmid selection in live vaccine vectors is needed.
Purpose of the Study:
- To develop and evaluate a novel plasmid selection system for live bacterial vaccine vectors based on immunity to the antimicrobial peptide microcin H47 (MccH47).
- To compare the immunogenicity of bacterial live vectors carrying plasmids with MccH47 immunity markers versus conventional ampicillin resistance markers.
- To assess the potential of heterologous prime-boost immunization strategies using these novel vectors.
Main Methods:
- Constructed two expression plasmids encoding green fluorescent protein (GFPuv): one with mchI for MccH47 immunity (pGEN222I) and one with bla for ampicillin resistance (pGEN222).
- Immunized mice intranasally with Salmonella enterica serovar Typhi CVD 908-htrA or Shigella flexneri 2a CVD 1208S live vectors carrying these plasmids.
- Analyzed GFPuv-specific serum immunoglobulin G (IgG) antibody responses after parenteral boosting with purified GFPuv.
- Compared homologous and heterologous prime-boost immunization regimens.
Main Results:
- Both pGEN222 and pGEN222I constructs elicited similar IgG responses when used with CVD 1208S or CVD 908-htrA(pGEN222I).
- CVD 908-htrA(pGEN222I) induced significantly higher IgG responses compared to CVD 908-htrA(pGEN222).
- Heterologous prime-boost immunization with two unrelated live vectors enhanced IgG responses compared to homologous priming with CVD 908-htrA(pGEN222I).
Conclusions:
- Microcin H47 (MccH47) immunity provides a viable and safer alternative for plasmid selection in bacterial live vectors, improving clinical acceptability.
- The success of heterologous priming strategies validates the potential for developing multivalent live vector-based vaccines against multiple human pathogens.
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