Choudary Sumit, Ravikumar P, Ashok Kumar C, Suryanarayana V V S, Reddy G R
Indian Veterinary Research Institute Campus, Hebbal, Bangalore, 560024 Karnataka, India.
Virus Genes. 2008 Aug;37(1):81-7. doi: 10.1007/s11262-008-0243-z. Epub 2008 May 31.
Foot and mouth disease (FMD) is the major constraint to international trade in livestock and animal products. Though conventional vaccine has shown to provide protection, it has several limitations, like short duration of immunity and poor cell mediated immune response compared to DNA vaccines, which are known to induce both cell mediated as well as humoral responses. The present work envisages the production of DNA vaccine construct with partial 1D gene (coding for VP1) of FMDV type 'A' and studied the efficacy of the vaccine coated on cationic PLGA micro-particles in guinea pigs. Sequence coding for VP1 of serotype 'A' was amplified by PCR and cloned into mammalian expression vector, pCDNA-containing FMDV IRES. Expression of the construct was confirmed by transfection of the plasmid into BHK-21 cells followed by the protein profile by SDS-PAGE and Western blotting of the cell lysate. Guinea pigs were immunized with 25 mug of the vaccine construct intramuscularly, followed by a booster at 21st day. Sera from the animals of all the groups (pre-vaccinated, 14, 21, and 28 days of post-vaccination) was analyzed by ELISA and SNT. ELISA titers indicated significant improvement in the antibody titers in the PLG-coated DNA group (2.408 + 0.06), whereas the naked plasmid gave a titer of 1.505+. Serum neutralization titers were higher in PLG-coated vaccine group compared to the animals that received the naked DNA vaccine. Increased CTL response measured by MTT stimulation index (1.58 + 0.08) was observed in the case of PLG-coated DNA vaccine construct compared to the naked DNA vaccine (1.29 + 0.068). PLG-DNA vaccine construct conferred 100% protection to the animals when challenged with 100GpID50 of homologous virus compared to 50% protection in case of naked DNA vaccine construct. The present study has shown that adjuvantation with PLG markedly improved the efficacy of DNA vaccine against FMDV.
口蹄疫(FMD)是家畜和畜产品国际贸易的主要制约因素。尽管传统疫苗已显示出能提供保护作用,但它有几个局限性,比如免疫持续时间短,与DNA疫苗相比细胞介导的免疫反应较差,而DNA疫苗已知能诱导细胞介导以及体液反应。目前的工作设想构建一种携带口蹄疫病毒A型部分1D基因(编码VP1)的DNA疫苗,并研究包被在阳离子聚乳酸-羟基乙酸共聚物(PLGA)微粒上的该疫苗在豚鼠体内的效力。通过聚合酶链反应(PCR)扩增血清型A的VP1编码序列,并克隆到含有口蹄疫病毒内部核糖体进入位点(IRES)的哺乳动物表达载体pCDNA中。通过将质粒转染到BHK-21细胞中,随后对细胞裂解物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹分析来确认构建体的表达。用25微克疫苗构建体对豚鼠进行肌肉注射免疫,然后在第21天进行加强免疫。通过酶联免疫吸附测定(ELISA)和血清中和试验(SNT)分析所有组(接种前、接种后14天、21天和28天)动物的血清。ELISA效价表明,PLG包被的DNA组(2.408 + 0.06)的抗体效价有显著提高,而裸质粒的效价为1.505 +。与接受裸DNA疫苗的动物相比,PLG包被的疫苗组的血清中和效价更高。与裸DNA疫苗(1.29 + 0.068)相比,在PLG包被的DNA疫苗构建体的情况下,通过噻唑蓝(MTT)刺激指数(1.58 + 0.08)测量的细胞毒性T淋巴细胞(CTL)反应增加。当用100个豚鼠感染剂量50(GpID50)的同源病毒攻击时,PLG-DNA疫苗构建体为动物提供了100%的保护,而裸DNA疫苗构建体的保护率为50%。本研究表明,用PLGA佐剂显著提高了DNA疫苗对口蹄疫病毒的效力。