猪繁殖与呼吸综合征病毒(PRRSV)导致生殖衰竭和呼吸道症状,给养猪业造成了巨大的经济损失。尽管市场上有几种针对PRRSV的疫苗,但它们显示出总体疗效低,一些国家需要覆盖当地的疫苗,循环变体。该项目旨在开发一种新的针对PRRSV特异性表位的嵌合抗原,并评估两种测试佐剂以配制疫苗候选物。测试抗原称为LTB-PRRSV,其在大肠杆菌中重组产生并且由来自大肠杆菌(LTB)的热不稳定肠毒素B亚基和来自PRRSV的四个表位组成。LTB-PRRSV被拯救为包涵体及其溶解方法,基于IMAC的纯化,重新折叠是标准化的,导致每升培养物平均产量为18毫克纯蛋白质。鉴于其生物相容性,层状双氢氧化物(LDH)已被用作疫苗佐剂,低成本,和正表面电荷,可以有效吸附带负电荷的生物分子。因此,选择LDH作为LTB-PRRSV的递送载体。通过在不同的LDH:LTB-PRRSV质量比(1:0.25、1:0.5、1:1和1:2)和pH9.5下孵育,将纯LTB-PRRSV吸附到LDH上。最佳吸附发生在1:2的质量比,和蔗糖-吐温溶液。获得的缀合物具有0.26的多分散指数,192nm的流体动力学直径,抗原终浓度为64.2μg/mL。通过向小鼠注射LDH:LTB-PRRSV,明矾/LTB-PRRSV,或LTB-PRRSV在包括以两周间隔和两个剂量水平(1和5μg)的三次免疫的方案中。LTB-PRRSV能够诱导强烈的体液反应,当LDH用作递送媒介物/佐剂时,其持续较长时间。讨论了LDH作为兽医疫苗有吸引力的载体的潜力。
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes reproductive failure and respiratory symptoms, leading to huge economic losses for the pig farming industry. Although several vaccines against PRRSV are available in the market; they show an overall low efficacy, and several countries have the need for vaccines covering the local, circulating variants. This project aims at developing a new chimeric antigen targeting specific epitopes from PRRSV and evaluating two test adjuvants to formulate a vaccine candidate. The test antigen was called LTB-PRRSV, which was produced recombinantly in Escherichia coli and consisted of the heat labile enterotoxin B subunit from E. coli (LTB) and four epitopes from PRRSV. LTB-PRRSV was rescued as inclusion bodies and methods for its solubilization, IMAC-based purification, and refolding were standardized, leading to mean yields of 18 mg of pure protein per liter culture. Layered double hydroxides (LDH) have been used as vaccine adjuvants given their biocompatibility, low cost, and positive surface charge that allows an efficient adsorption of negatively charged biomolecules. Therefore, LDH were selected as delivery vehicles of LTB-PRRSV. Pure LTB-PRRSV was adsorbed onto LDH by incubation at different LDH:LTB-PRRSV mass ratios (1:0.25, 1:0.5, 1:1, and 1:2) and at pH 9.5. The best adsorption occurred with a 1:2 mass ratio, and in a sucrose-tween solution. The conjugates obtained had a polydispersity index of 0.26, a hydrodynamic diameter of 192 nm, and a final antigen concentration of 64.2 μg/mL. An immunogenicity assessment was performed by injecting mice with LDH:LTB-PRRSV, Alum/LTB-PRRSV, or LTB-PRRSV in a scheme comprising three immunizations at two-week intervals and two dose levels (1 and 5 μg). LTB-PRRSV was capable of inducing strong humoral responses, which lasted for a longer period when LDH was used as the delivery vehicle/adjuvant. The potential of LDH to serve as an attractive carrier for veterinary vaccines is discussed.