Park Yong Sung, Lee Jin Hyup, Hung Chien-Fu, Wu T-C, Kim Tae Woo
Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Seoul, South Korea.
Infect Immun. 2008 May;76(5):1952-9. doi: 10.1128/IAI.01722-07. Epub 2008 Feb 19.
The generation of protective humoral immune responses against the receptor-binding domain (domain IV) of protective antigen [PA(dIV)] of Bacillus anthracis represents a plausible approach against anthrax toxin. In the current study, we have developed a naked DNA vaccine encoding calreticulin (CRT) linked to PA(dIV) of Bacillus anthracis [CRT/PA(dIV)]. We transfected a human embryonic kidney cell line (HEK 293) with CRT/PA(dIV) DNA and performed Western blotting and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the endoplasmic reticulum, resulting in secretion of the chimeric CRT/PA(dIV) protein. We then evaluated the ability of CRT/PA(dIV) DNA to generate PA(dIV)-specific antibody responses and protective immunity against lethal anthrax toxin (PA plus lethal factor) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses than mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4 dependent, since CD4 knockout mice demonstrated a significant reduction in antibody responses. In addition, analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT/PA(dIV) DNA vaccine accelerated the avidity maturation of antibodies to PA(dIV) compared to vaccination with PA(dIV) DNA. Importantly, the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge. Thus, DNA vaccines encoding CRT linked to PA(dIV) may dramatically enhance PA-specific protective antibody responses. Our results have significant clinical applications for biodefense against anthrax toxin.
针对炭疽杆菌保护性抗原[PA(dIV)]的受体结合结构域（结构域IV）产生保护性体液免疫反应是一种对付炭疽毒素的可行方法。在本研究中，我们开发了一种编码与炭疽杆菌PA(dIV)相连的钙网蛋白（CRT）的裸DNA疫苗[CRT/PA(dIV)]。我们用CRT/PA(dIV) DNA转染了人胚肾细胞系（HEK 293），并进行了蛋白质免疫印迹和共聚焦显微镜分析。我们发现，CRT与PA(dIV)的连接将PA(dIV)靶向至内质网，导致嵌合的CRT/PA(dIV)蛋白分泌。然后，我们评估了CRT/PA(dIV) DNA产生PA(dIV)特异性抗体反应以及针对致死性炭疽毒素（PA加致死因子）攻击的保护性免疫的能力。我们发现，用CRT/PA(dIV) DNA免疫的小鼠比单独用PA(dIV) DNA免疫的小鼠能够更快地诱导出显著更高的PA(dIV)特异性抗体反应。此外我们观察到，由CRT/PA(dIV) DNA产生的这种增强的抗体反应是CD4依赖性的，因为CD4基因敲除小鼠的抗体反应显著降低。另外，对诱导产生的PA特异性抗体的效价和亲和力成熟度分析表明，与用PA(dIV) DNA疫苗接种相比，用CRT/PA(dIV) DNA疫苗接种加速了抗体对PA(dIV)的亲和力成熟。重要的是，增强的抗体反应与针对致死性炭疽毒素攻击的保护性免疫相关。因此，编码与PA(dIV)相连的CRT的DNA疫苗可能会显著增强PA特异性保护性抗体反应。我们的结果在针对炭疽毒素的生物防御方面具有重要的临床应用价值。
