Hunter Joseph G L, Wilde Shyra, Tafoya Amanda M, Horsman Jamie, Yousif Miranda, Diamos Andrew G, Roland Kenneth L, Mason Hugh S
Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
School of Life Sciences, Arizona State University, Tempe, AZ, USA.
PeerJ. 2019 Mar 28;7:e6600. doi: 10.7717/peerj.6600. eCollection 2019.
Necrotic enteritis (NE) is caused by type A strains of the bacterium . Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE.
We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds.
Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.
坏死性肠炎（NE）由A型细菌菌株引起。据估计，全球家禽业因坏死性肠炎每年的经济损失超过20亿美元。传统上，通过在禽类饲料中添加抗生素可有效控制坏死性肠炎。然而，最近对这种做法对人类病原体抗生素耐药性增加的影响的担忧促使我们考虑采用替代方法，如疫苗接种，来控制这种疾病。坏死性肠炎菌株产生两种主要毒素，α毒素和NetB。针对任何一种毒素的免疫反应都可以提供对坏死性肠炎的部分保护。
我们开发了一种融合蛋白，该融合蛋白结合了α毒素的无毒羧基末端结构域（PlcC）和NetB的减毒突变形式（NetB-W262A），用作疫苗抗原来免疫禽类以预防坏死性肠炎。我们利用了一个经过密码子优化的DNA序列以实现高水平表达。6-组氨酸标记的PlcC-NetB融合蛋白在植物中使用双生病毒复制子瞬时表达系统合成，通过金属亲和层析纯化，并用于免疫肉鸡。
免疫的禽类对植物产生的PlcC-NetB蛋白以及细菌产生的His-PlcC和His-NetB均产生强烈的血清IgY反应。当用融合蛋白处理时，免疫的禽类在随后接受强毒细菌的饲料攻毒时得到了显著保护。这些结果表明，植物产生的PlcC-NetB类毒素是控制家禽坏死性肠炎的一种有前景的疫苗候选物。
