State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; MARA Key Laboratory of Development of Veterinary Diagnostic Products, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.
Department of Microbiology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Vaccine. 2022 Jun 15;40(27):3771-3780. doi: 10.1016/j.vaccine.2022.05.021. Epub 2022 May 20.
Dermonecrotic toxin (DNT) is an important bacterial virulence factor produced by the zoonotic pathogens Bordetella bronchiseptica and Pasteurella multocida. This study aims to explore the possibility of expressing different fragments of P. multocida toxin (PMT) in the chromosome of attenuated B. bronchiseptica to generate single-component mucosal vaccine candidates. To achieve this, a 954-bp fragment (basepairs 301 ∼ 1254) of the B. bronchiseptica aroA gene was replaced with an N-terminal, 930-bp fragment (basepairs 1-930; PMTN) or a C-terminal, 900-bp fragment (base pairs 2959 ∼ 3858; PMTC) of the PMT encoding gene toxA. The resulting strains, denoted as Bb-PMTN or Bb-PMTC, expressed PMTN and PMTC, as evidenced by ELISA using polyclonal against full-length of PMT. Phenotypical analyses revealed that Bb-PMTN and Bb-PMTC grew much slower than wild type strains in tryptic soy broth. These strains also displayed significantly decreased 161-fold-virulence compared to the wildtype strains in mouse models. Intranasal immunization of Bb-PMTN and Bb-PMTC in mice induced high levels of antibodies against B. bronchiseptica and PMT, as well as IFN-γ and IL-10 in mouse sera, and most importantly, high titers of sIgA in mouse lungs. Vaccination with these two engineering strains provided 100% protection of mice against lethal challenge with B. bronchiseptica and 80%∼100% protection against lethal challenge with PMT, with Bb-PMTN exhibiting 1.25-fold greater immunogenic efficacy over Bb-PMTC. This study highlights the use of B. bronchiseptica attenuated strains as live mucosal vectors to deliver heterologous antigens.
坏死毒素（DNT）是一种重要的细菌毒力因子，由人畜共患病病原体支气管败血波氏杆菌和多杀性巴氏杆菌产生。本研究旨在探索在减毒支气管败血波氏杆菌染色体中表达不同片段的巴氏杆菌毒素（PMT）的可能性，以生成单组分粘膜疫苗候选物。为此，用一个 954 个碱基对（碱基对 301∼1254）的支气管败血波氏杆菌 aroA 基因替换 N 端的 930 个碱基对（碱基对 1-930；PMTN）或 C 端的 900 个碱基对（碱基对 2959∼3858；PMTC）的 PMT 编码基因 toxA。结果表明，Bb-PMTN 和 Bb-PMTC 表达了 PMTN 和 PMTC，这可以通过 ELISA 检测多克隆抗体针对全长 PMT 来证明。表型分析显示，与野生型菌株相比，Bb-PMTN 和 Bb-PMTC 在胰蛋白酶大豆肉汤中生长速度明显较慢。这些菌株在小鼠模型中的毒力也显著降低了 161 倍。在小鼠中进行鼻内免疫 Bb-PMTN 和 Bb-PMTC 可诱导针对支气管败血波氏杆菌和 PMT 的高抗体水平，以及血清中的 IFN-γ和 IL-10，最重要的是，在小鼠肺部产生高滴度的 sIgA。用这两种工程菌株接种可使小鼠对致死性支气管败血波氏杆菌攻击的保护率达到 100%，对致死性 PMT 攻击的保护率达到 80%∼100%，其中 Bb-PMTN 的免疫原性比 Bb-PMTC 高 1.25 倍。本研究强调了利用减毒支气管败血波氏杆菌作为活的粘膜载体传递异源抗原。
