PDF(Pubmed)
Abstract：
Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.
摘要：
猪流行性腹泻病毒（PEDV）引起的猪流行性腹泻（PED），是一种急性和高度传染性疾病，给养猪业造成了巨大的经济损失。鉴于PEDV主要感染肠道的粘膜表面，提高粘膜免疫对防止病毒入侵至关重要。乳酸菌（LAB）口服疫苗在对抗粘膜感染性疾病方面具有独特的优势和潜在的应用。使它们成为控制PED爆发的理想方法。然而,传统的LAB口服疫苗使用质粒进行外源蛋白表达和抗生素基因作为选择标记。抗生素基因可以通过转座扩散，转让,或同源重组，导致耐药菌株的产生。为了克服这些问题,已经开发了基因组编辑技术来实现LAB基因组中的基因表达。在这项研究中,我们使用CRISPR-NCas9系统将PEDVS1基因整合到丙氨酸消旋酶缺陷型副干酪乳杆菌△AlrHLJ-27的基因组中(L.副干酪△AlrHLJ-27）在胸苷酸合成酶（thyA）位点，产生应变，S1/△AlrHLJ-27。我们在小鼠和仔猪中进行了免疫试验，以评估免疫反应水平，并通过仔猪攻击试验评估其对PEDV的保护作用。小鼠和仔猪口服S1/△AlrHLJ-27菌株引起粘膜，体液,和细胞免疫反应。该菌株在仔猪中对PEDV感染也表现出一定水平的抗性。这些结果证明了S1/△AlrHLJ-27作为PEDV控制的口服疫苗候选物的潜力。关键点:•构建菌株S1/△AlrHLJ-27作为口服疫苗的候选物。•进行免疫原性应答和攻击测试以分析菌株的能力。•菌株S1/△AlrHLJ-27可以在一定程度上保护仔猪。
