Souod Negar, Kargar Mohammad, Hoseini Mohammad Hossein, Jafarinia Mojtaba
Department of Genetics, Marvdasht Branch, Islamic Azad University, Marvdasht, I. R, Iran.
Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, I.R, Iran.
Microb Pathog. 2021 Aug;157:104978. doi: 10.1016/j.micpath.2021.104978. Epub 2021 May 19.
Development of an effective oral vaccine against Cholera, a life-threatening dehydrating diarrheal disease, proved to be a challenging task. To improve oral subunit vaccine immunogenicity and to prevent the state of oral tolerance, application of mucosal adjuvants might be a promising approach. In the present study, the CtxB-TcpA-C-CPE fusion was constructed in which CtxB and C-CPE were used as mucosal adjuvants and vaccine delivery system, respectively, to induce mucosal immune responses, and to improve the anti-toxin and anti-colonizing immunity against V. cholerae.
MATERIALS & METHODS: The fusion construct was synthesized, sub-cloned in pQE30 and expressed in E. coli. The three antigen, making the fusion protein, were also separately expressed in E. coli. The recombinant proteins were purified by affinity chromatography using Ni-NTA agarose. Western blot analysis using anti-His antibody was applied to confirm identity of the purified proteins. BALB/c mice were subcutaneously immunized with CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE separately. The mice were orally immunized (in 3 boosts) by the same vaccine. Mucosal immune response stimulation was evaluated by measuring the levels of intestinal IgA. Systemic immune response was evaluated by measuring total serum IgG, IgG1, IgG2a, IgG2b subclasses, and also IL-4, IL-5, IL-10 and IFN-γ cytokines in spleen cell culture.
The recombinant proteins CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE were expressed in E. coli and highly purified in a single step of chromatography. BALB/c mice immunized with the fusion protein had highest levels of intestinal IgA, serum IgG and IgG subclasses, compared to each of the three proteins making the fusion. Moreover, stimulated splenocytes of mice immunized with the fusion protein displayed significantly higher amounts of IL-5 and IFN-ɣ cytokines. Th2 dominance of the immune response was more evident in mice receiving the fusion protein.
Inclusion of CtxB, as the mucosal adjuvant, and C-CPE, as the vaccine delivery system, in the fusion protein CtxB-TcpA-C-CPE significantly enhanced the elicited mucosal and systemic immune responses, compared to TcpA alone. Of note, significant production of intestinal IgA in mice immunized with the fusion protein is presumably capable of neutralizing TcpA, CtxB and C-CPE antigens, preventing V. cholera colonization, and toxic function of CtxB and C-CPE. Challenge infection of the immunized mice is required to evaluate protective potential of the fusion protein against V. cholera.
霍乱是一种危及生命的导致脱水的腹泻疾病，开发一种有效的霍乱口服疫苗是一项具有挑战性的任务。为提高口服亚单位疫苗的免疫原性并防止口服耐受状态，应用黏膜佐剂可能是一种有前景的方法。在本研究中，构建了CtxB-TcpA-C-CPE融合蛋白，其中CtxB和C-CPE分别用作黏膜佐剂和疫苗递送系统，以诱导黏膜免疫反应，并增强针对霍乱弧菌的抗毒素和抗定植免疫力。
合成融合构建体，亚克隆至pQE30并在大肠杆菌中表达。构成融合蛋白的三种抗原也分别在大肠杆菌中表达。重组蛋白通过使用Ni-NTA琼脂糖的亲和层析进行纯化。应用抗His抗体的蛋白质免疫印迹分析来确认纯化蛋白的身份。将BALB/c小鼠分别皮下免疫CtxB、TcpA、C-CPE和融合蛋白CtxB-TcpA-C-CPE。小鼠通过相同疫苗进行口服免疫（3次加强免疫）。通过测量肠道IgA水平评估黏膜免疫反应刺激。通过测量血清总IgG、IgG1、IgG2a、IgG2b亚类以及脾细胞培养中的IL-4、IL-5、IL-10和IFN-γ细胞因子评估全身免疫反应。
重组蛋白CtxB、TcpA、C-CPE和融合蛋白CtxB-TcpA-C-CPE在大肠杆菌中表达，并在一步层析中高度纯化。与构成融合蛋白的三种蛋白中的每一种相比，用融合蛋白免疫的BALB/c小鼠具有最高水平的肠道IgA、血清IgG和IgG亚类。此外，用融合蛋白免疫的小鼠的受刺激脾细胞显示出显著更高量的IL-5和IFN-γ细胞因子。在接受融合蛋白的小鼠中，免疫反应的Th2优势更为明显。
与单独的TcpA相比，在融合蛋白CtxB-TcpA-C-CPE中包含作为黏膜佐剂的CtxB和作为疫苗递送系统的C-CPE可显著增强诱导的黏膜和全身免疫反应。值得注意的是，用融合蛋白免疫的小鼠中肠道IgA的大量产生可能能够中和TcpA、CtxB和C-CPE抗原，防止霍乱弧菌定植以及CtxB和C-CPE的毒性作用。需要对免疫小鼠进行攻毒感染以评估融合蛋白对霍乱弧菌的保护潜力。
