Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Vaccine. 2011 Jun 15;29(27):4521-33. doi: 10.1016/j.vaccine.2011.03.082. Epub 2011 Apr 17.
The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in highlighting the efficacy of plant expressed PA(dIV) by oral immunization in murine model.
现有的炭疽疫苗存在不完全特征、潜在的致反应性和扩大剂量方案等局限性。植物疫苗为疫苗生产提供了安全的替代选择。在本研究中，我们在植物体内（通过核农杆菌和叶绿体转化）和大肠杆菌中表达了炭疽芽孢杆菌保护性抗原基因 [PA(dIV)] 的结构域 IV [rPA(dIV)]。通过分子分析证实了转基因的存在和 PA(dIV)在植物体内的表达。在叶绿体转化体中，富含 AT（71.8% AT 含量）的 PA(dIV)基因表达水平高达总可溶性蛋白 (TSP) 的 5.3%，而在核转化体中则获得了 0.8%的 TSP。此外，我们通过腹腔内（i.p.）和口服免疫接种，研究了植物和大肠杆菌来源的 PA(dIV)在小鼠中的保护反应，同时使用或不使用佐剂。腹腔内和口服免疫接种植物来源的 PA(dIV)后，抗体滴度均>10(4)，口服免疫接种大肠杆菌来源的 PA(dIV)后，抗体滴度>10(4)。用佐剂处理的大肠杆菌来源的 PA(dIV)腹腔内注射可产生>10(5)的最高抗体滴度。所有免疫组均表现出 IgG1 滴度显著高于 IgG2a，表明存在极化的 Th2 型反应。我们还评估了口服免疫组的粘膜抗体反应。当分析粪便提取物时，在佐剂处理的植物和大肠杆菌来源的 PA(dIV)组中，低 sIgA 滴度被证明。此外，PA(dIV)抗血清在体外增强了巨噬细胞对炭疽芽孢杆菌孢子的摄取，并且还表现出抗发芽作用，这表明其在粘膜表面具有强大的作用。来自各种组的抗体在体外有效地中和了致死毒素。当用炭疽芽孢杆菌攻击小鼠时，用佐剂处理的植物 PA(dIV)免疫的小鼠分别赋予 60%和 40%的保护，而大肠杆菌来源的 PA(dIV)经腹腔内和口服免疫接种分别赋予 100%和 80%的保护。因此，我们的研究首次尝试在小鼠模型中强调口服免疫接种植物表达的 PA(dIV)的功效。
