ABSTRACT

The proficiency of Salmonella Typhi to induce cell-mediated immunity has allowed its exploitation as a live vector against the obligate intracellular protozoan Toxoplasma gondii. T. gondii vaccine research is of great medical value due to the lack of a suitable toxoplasmosis vaccine. In the present work, we integrated T. gondii antigen into a growth-dependent chromosome locus guaBA of S. Typhi CVD910 strain to form recombinant S. Typhi monovalent CVD910-SAG1 expressed T. gondii SAG1 antigen and monovalent CVD910-GRA2 expressed T. gondii GRA2 antigen. Furthermore, a low-copy stabilized recombinant plasmid encoding SAG1 antigen was transformed into CVD910-GRA2 to form bivalent CVD910-GS strain. An osmolarity-regulated promoter was also incorporated to control the gene transcription, whereas clyA export protein was included to translocate the antigen out of the cytoplasm. Both CVD910-GRA2 and CVD910-GS displayed healthy growth fitness and readily expressed the encoded T. gondii antigens. When administered in vivo, CVD910-GS successfully induced both humoral and cellular immunity in the immunized BALB/c mice, and extended mice survival against virulent T. gondii. In particular, the mice immunized with bivalent CVD910-GS presented the highest titers of IgG, percentages of CD4⁺ T, CD8⁺ T, B cells and memory T cells, and total IgG⁺ memory B cells as compared to the CVD910-GRA2 and control strains. The CVD910-GS group also generated mixed Th1/Th2 cytokine profile with secretions of IFN-ɣ, IL-2 and IL-10. This study demonstrated the importance of enhancing live vector fitness to sustain heterologous antigen expression for eliciting robust immune responses and providing effective protection against pathogen.

摘要

伤寒沙门氏菌对诱导细胞介导免疫的能力使其能够作为活载体来对抗专性细胞内原生动物弓形虫。由于缺乏合适的弓形虫病疫苗，弓形虫疫苗研究具有很大的医学价值。在目前的工作中，我们将T. gondii抗原整合到S的生长依赖性染色体基因座guaBA中。伤寒CVD910菌株形成重组沙门氏菌。伤寒单价 CVD910-SAG1 表达弓形虫SAG1 抗原，单价 CVD910-GRA2 表达弓形虫GRA2抗原。此外，将编码SAG1抗原的低拷贝稳定重组质粒转化到CVD910-GRA2中，形成二价CVD910-GS菌株。还掺入了一个渗透压调节的启动子以控制基因转录，而包含 clyA 输出蛋白以将抗原转移出细胞质。CVD910-GRA2 和 CVD910-GS 都表现出健康的生长适应性，并且很容易表达编码的弓形虫抗原。在体内给药时，CVD910-GS 在免疫的 BALB/c 小鼠中成功地诱导了体液免疫和细胞免疫，并延长了小鼠对抗毒性弓形虫的存活时间。特别是，用二价 CVD910-GS 免疫的小鼠表现出最高的 IgG 滴度，CD4 ^+百分比与 CVD910-GRA2 和对照菌株相比，T、CD8 ⁺ T、B 细胞和记忆 T 细胞，以及总 IgG ^{+记忆 B 细胞。}CVD910-GS 组还产生混合的 Th1/Th2 细胞因子谱以及 IFN-ɣ、IL-2 和 IL-10 的分泌物。该研究证明了增强活载体适应性以维持异源抗原表达以引发强大的免疫反应并提供针对病原体的有效保护的重要性。