Center for Integrative Microbiology and Evolution, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway.
Department of Clinical Microbiology, Clinical Bacteriology, and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå, Sweden.
Vaccine. 2017 Dec 19;35(52):7264-7272. doi: 10.1016/j.vaccine.2017.11.009. Epub 2017 Nov 16.
Francisella noatunensis ssp. noatunensis (F.n.n.) is the causative agent of francisellosis in Atlantic cod and constitutes one of the main challenges for future aquaculture on this species. A facultative intracellular bacterium like F.n.n. exert an immunologic challenge against which live attenuated vaccines in general are most effective. Thus, we constructed a deletion in the F.n.n. clpB gene as ΔclpB mutants are among the most promising vaccine candidates in human pathogenic Francisella.
Characterization of F.n.n. ΔclpB using primary Atlantic cod head kidney leukocytes, the zebrafish embryo and adult zebrafish model with focus on potential attenuation, relevant immune responses and immunogenic potential.
Interleukin 1 beta transcription in Atlantic cod leukocytes was significantly elevated from 24 to 96 h post infection with F.n.n. ΔclpB compared to F.n.n. wild-type (wt). Growth attenuation of the deletion mutant in zebrafish embryos was observed by fluorescence microscopy and confirmed by genome quantification by qPCR. In the immunization experiment, adult zebrafish were immunized with 7 × 10 CFU of F.n.n. ΔclpB before challenge four weeks later with 6 × 10 CFU of F.n.n. wt. One day after challenge, immunized zebrafish responded with significantly lower interleukin 8 levels compared to the non-immunized control. Immunized fish were protected against the acute mortality observed in non-immunized zebrafish after challenge and bacterial genomes quantified by qPCR were reduced to a minimum 28 days post challenge, indicating protective immunity stimulated by F.n.n. ΔclpB.
Deletion mutation of clpB in F.n.n. causes in vitro and in vivo attenuation and elicits a protective immune response in adult zebrafish against a lethal dose of F.n.n. wt. Taken together, the results presented increases the knowledge on protective immune responses against F.n.n.
弗朗西斯氏菌亚种无土亚种（F.n.n.）是大西洋鳕鱼弗朗西斯氏菌病的病原体，是未来该物种水产养殖的主要挑战之一。像 F.n.n.这样的兼性细胞内细菌会对机体造成免疫挑战，而活减毒疫苗通常对此最有效。因此，我们构建了 F.n.n. clpB 基因的缺失突变体，因为 F.n.n. clpB 缺失突变体是人类致病性弗朗西斯氏菌最有前途的疫苗候选物之一。
利用大西洋鳕鱼头肾白细胞、斑马鱼胚胎和成年斑马鱼模型来描述 F.n.n.ΔclpB 的特性，重点关注其潜在的减毒作用、相关免疫反应和免疫原性。
与 F.n.n.野生型（wt）相比，F.n.n.ΔclpB 感染大西洋鳕鱼白细胞后，白细胞中白细胞介素 1β的转录从 24 小时到 96 小时显著升高。通过荧光显微镜观察到缺失突变体在斑马鱼胚胎中的生长衰减，并通过 qPCR 对基因组定量进行了确认。在免疫实验中，成年斑马鱼用 7×10 CFU 的 F.n.n.ΔclpB 免疫，四周后用 6×10 CFU 的 F.n.n. wt 进行攻毒。攻毒后 1 天，与未免疫对照组相比，免疫斑马鱼的白细胞介素 8 水平显著降低。与未免疫的斑马鱼相比，免疫鱼受到了保护，免受攻毒后急性死亡率的影响，攻毒后 28 天 qPCR 定量的细菌基因组减少到最低水平，表明 F.n.n.ΔclpB 刺激了保护性免疫。
F.n.n. clpB 的缺失突变导致体外和体内衰减，并在成年斑马鱼中引发针对致死剂量的 F.n.n. wt 的保护性免疫反应。总之，这些结果增加了对 F.n.n.的保护性免疫反应的了解。
