Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Vet Med Sci. 2023 Jul;9(4):1908-1922. doi: 10.1002/vms3.1173. Epub 2023 Jun 5.
Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen.
Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free-pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti-omp25-specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25-specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real-time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain.
Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the phosphate-buffered saline control group). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups had significantly greater IL-4 and IL-10 transcription levels than the other groups. The spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process.
Our study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost-effective, safe, sustainable, simple pilot development of pharmaceutical products.
大多数布鲁氏菌感染发生在黏膜上。因此，开发通过黏膜递送的疫苗对于控制布鲁氏菌病可能至关重要。因此，我们评估了基于乳球菌 lactis 的重组口服抗原递送系统对布鲁氏菌 abortus omp25 抗原的功效。
用 pNZ8148 变体（编码 omp25 的 pNZ8148:omp25 和游离 pNZ8148）转化的乳球菌 lactis 对小鼠进行口服免疫接种。在动物组免疫接种 30 天后，通过 ELISA 试验评估抗 omp25 特异性 IgG1 抗体。此外，通过 ELISA 分析含有 omp25 特异性分泌型 IgA (sIgA) 的鼻和支气管肺泡灌洗液。ELISA 试验和实时 PCR 也用于分析最后一次加强免疫后 28 天内的细胞因子反应。此外，通过使 BALB/c 小鼠接触布鲁氏菌 abortus 菌株，评估了 L. lactis pNZ8148:omp25 疫苗的保护潜力。
根据初步筛选结果，确定 omp25 蛋白具有免疫原性，因为它具有最大的可溶性和柔韧性以及 0.75 的抗原性值。产生的质粒用 KpnI 和 XbaI 消化。通过电泳分离 786 bp 的消化片段，确认成功生产了重组质粒。在蛋白质水平上的抗原表达表明，目标组产生了 25 kDa 大小的 omp25 蛋白，但对照组没有蛋白表达。在初次免疫后 14 天，用 pNZ8148-Usp45-omp25-L. lactis 免疫的小鼠血清中存在大量的 omp25 特异性 IgG1（与磷酸盐缓冲盐水对照组相比，目标组 p < 0.001）。在 pNZ8148-Usp45-omp25-L. lactis 和 IRBA 免疫接种的小鼠样本中，IFN-γ 和 TNF-α 水平在第 14 天和第 28 天分别更为显著（p < 0.001）。与其他组相比，pNZ8148-Usp45-omp25-L. lactis 和 IRBA 免疫接种组的 IL-4 和 IL-10 转录水平显著更高。与其他组相比，pNZ8148-Usp45-omp25-L. lactis 和 IRIBA 疫苗组的脾部分脾损伤、肺泡水肿、淋巴细胞浸润和炎症过程引起的形态损伤程度较低。
我们的研究提供了一种新的方法，利用食品级、非致病性和非商业性细菌乳球菌 lactis 作为蛋白质细胞工厂来生产新型免疫原性融合候选物 romp25。该方法为评估具有成本效益、安全、可持续、简单的药物产品试点开发提供了一种有吸引力的新方法。
