Webb Robert P, Smith Theresa J, Wright Patrick, Brown Jennifer, Smith Leonard A
United States Army Medical Research Institute for Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, United States.
Vaccine. 2009 Jul 16;27(33):4490-7. doi: 10.1016/j.vaccine.2009.05.030. Epub 2009 May 28.
A recombinant, catalytically inactive Clostridium botulinum neurotoxin A1 holoprotein (ciBoNT/A1 HP) was constructed by introducing amino acid substitutions H223A, E224A, and H227A in the active site to ablate proteolytic activity. ciBoNT/A1 HP was produced in the yeast Pichia pastoris and the purified product was evaluated as a vaccine candidate by comparison against recombinant BoNT/A1 LC, LC-belt, LC-H(n), and H(c) antigens and a LC-H(n)+H(c) combination in mouse potency and efficacy bioassays when challenged with BoNT/A subtypes /A1, /A2, and /A3. A single dose of ciBoNT/A1 HP provided equivalent or greater protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence. Only the LC-H(n)+H(c) combination provided comparable protection against /A1; however, it was less effective against subtypes /A2 and /A3. Differences in protective immunity diminished after multiple vaccinations with either ciBoNT/A1 HP or BoNT/A1 H(c), and the survival rates were more comparable at the toxin levels used to challenge.
通过在活性位点引入氨基酸取代H223A、E224A和H227A以消除蛋白水解活性，构建了一种重组的、催化无活性的肉毒杆菌神经毒素A1全蛋白（ciBoNT/A1 HP）。ciBoNT/A1 HP在毕赤酵母中产生，当用BoNT/A亚型/A1、/A2和/A3攻击时，通过在小鼠效力和功效生物测定中与重组BoNT/A1轻链（LC）、LC-带、LC-H(n)和H(c)抗原以及LC-H(n)+H(c)组合进行比较，对纯化产物作为疫苗候选物进行评估。单剂量的ciBoNT/A1 HP不仅对同源毒素，而且对具有显著氨基酸差异的两种不同毒素亚型提供了同等或更高的保护性免疫。只有LC-H(n)+H(c)组合对/A1提供了相当的保护；然而，它对/A2和/A3亚型的效果较差。用ciBoNT/A1 HP或BoNT/A1 H(c)多次接种后，保护性免疫的差异减小，并且在用于攻击的毒素水平下存活率更具可比性。
