Fang Chee-Mun, Wang Jin Yuan, Chinchilla Magaly, Levine Myron M, Blackwelder William C, Galen James E
Department of Medicine, Division of Geographic Medicine, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Infect Immun. 2008 Oct;76(10):4422-30. doi: 10.1128/IAI.00487-08. Epub 2008 Jul 28.
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.
表达异源抗原的减毒活细菌菌株是一种有吸引力的疫苗开发策略。然而，使用耐药基因来选择导入活载体的表达质粒存在理论上的健康风险。因此，我们开发了一种基于对抗菌肽微菌素H47（MccH47）免疫的质粒选择新方法。构建了两个编码报告绿色荧光蛋白（GFPuv）的表达质粒；选择标记要么是赋予对MccH47免疫的mchI（pGEN222I），要么是赋予对氨苄青霉素常规抗性的bla（编码β-内酰胺酶）（pGEN222）。在用肠炎沙门氏菌血清型伤寒杆菌CVD 908-htrA或福氏志贺氏菌2a CVD 1208S活载体经鼻内免疫并用纯化的GFPuv进行肠胃外加强免疫的小鼠中，分析了GFPuv特异性血清免疫球蛋白G（IgG）抗体反应。当使用CVD 1208S或CVD 908-htrA（pGEN222I）作为载体时，观察到pGEN222和pGEN222I的IgG抗体反应相似。有趣的是，CVD 908-htrA（pGEN222I）引发的IgG反应明显高于CVD 908-htrA（pGEN222）。我们还比较了用CVD 908-htrA（pGEN222I）或CVD 1208S（pGEN222I）进行同源启动与先用CVD 908-htrA（pGEN222I）然后用CVD 1208S（pGEN222I）进行异源启动以及反之亦然的启动潜力。与同源CVD 908-htrA（pGEN222I）产生的反应相比，用两种不相关的活载体免疫显著增强了IgG反应，但与CVD 1208S（pGEN222I）的反应相比则不然。MccH47为细菌活载体中的质粒选择提供了一种替代系统，大大提高了它们的临床可接受性。此外，异源启动策略的成功支持了未来开发针对多种人类病原体的多价活载体免疫策略的可行性。
