Infection and Immunity Program, Monash Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, Victoria, Australia.
CSIRO Biosecurity Flagship, Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Appl Environ Microbiol. 2021 May 11;87(11). doi: 10.1128/AEM.00392-21.
Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate La3 (previously ) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, La3::P -r and La3::P -rB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens. Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of La3 as a live delivery vehicle for recombinant proteins.
动物生产系统中的疾病控制需要持续警惕。从历史上看，应用饲料抗生素来控制细菌和提高性能一直是维持动物健康和福利的常用方法。然而，广泛使用饲料抗生素被认为会增加抗生素耐药性发展的风险。需要开发替代方法来控制疾病和维持生产力。活疫苗接种可通过诱导粘膜免疫反应来有效预防粘膜定植病原体。本土家禽分离株 La3（以前称为）已被确定为用作活载体的候选物，以将治疗性蛋白质（如细菌素、噬菌体内溶素或疫苗抗原）递送到鸡的胃肠道中。在这项研究中，确定了 La3 的完整基因组序列，并进行了转录组分析以鉴定高表达基因。从组成型表达基因的预测启动子区域和核糖体结合位点构建重组蛋白表达盒。构建了一系列双交换穿梭质粒，以通过同源重组方便地将表达盒快速选择性整合到 La3 染色体中。在没有选择的情况下，插入物经过 100 代显示出 100%的稳定整合。发现蛋白表达水平与预测启动子强度之间存在正相关关系。使用该系统，在没有选择的情况下，成功地展示了抗原 rNetB 的稳定染色体表达。最后，构建并表征了两个重组菌株 La3::P-r 和 La3::P-rB，它们显示出作为鸡活疫苗的潜在应用前景。治疗性蛋白质（如抗原）可用于预防家禽的传染病。然而，通过肌肉内或皮下注射传统疫苗接种通常对生活在胃肠道内的粘膜定植微生物无效。利用活细菌将疫苗抗原直接递送到肠道免疫系统可以克服传统疫苗接种的一些局限性。在这项工作中，特别有效的肠道定植菌 La3 经过分析和工程化，具有模块化和稳定的表达系统，可产生重组蛋白。为了证明该系统的有效性，在没有选择的情况下监测了禽病原体抗原的表达超过 100 代，发现完全稳定。这项研究展示了遗传工具和新型组成型表达系统的开发，并进一步开发了 La3 作为重组蛋白的活递药载体。
