Hur Jin, Lee John Hwa
College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, South Korea.
Clin Vaccine Immunol. 2011 Feb;18(2):203-9. doi: 10.1128/CVI.00407-10. Epub 2010 Dec 15.
A plasmid harboring eltB, the gene encoding heat-labile enterotoxin (LTB), was constructed by insertion of eltB into an Asd(+) β-lactamase signal plasmid (pMMP65). This was introduced into the Δlon ΔcpxR Δasd Salmonella enterica serovar Typhimurium strain and designated the LTB adjuvant strain. LTB protein production and secretion from the strain were demonstrated with an immunoblot assay and enzyme-linked immunosorbent assay. The LTB strain was evaluated for enhancement of immunity and protection efficacy induced by a previously constructed live Salmonella vaccine candidate. In addition, immunization strategies using the LTB strain were optimized for effective salmonellosis protection. Seventy female BALB/c mice were divided into seven groups (A to G; n = 10 mice per group). Mice were primed at 6 weeks of age and boosted at 9 weeks of age. All mice were orally challenged with a virulent wild-type strain at week 3 postbooster. Serum IgG and IgA titers from mice immunized with the LTB strain alone or with a mixture of the LTB strain and the vaccine candidate were significantly increased. The secretory IgA titers from mice immunized with the LTB strain alone or with the mixture were at least 2.2 times greater than those of control mice. In addition, all group E mice (primed with the vaccine-LTB mixture and boosted with the vaccine candidate) were free of clinical signs of salmonellosis and survived a virulent challenge. In contrast, death due to the challenge was 100% in control mice, 80% in group A mice (single immunization with the vaccine candidate), 60% in group B mice (primed and boosted with the vaccine candidate), 40% in group C mice (single immunization with the LTB strain), 30% in group D mice (primed and boosted with the LTB strain), and 30% in group F mice (primed and boosted with the vaccine-LTB mixture). These results suggest that vaccination with the LTB strain, especially when added at the prime stage only, effectively enhances immune responses and protection against salmonellosis.
通过将编码不耐热肠毒素（LTB）的eltB基因插入Asd(+)β-内酰胺酶信号质粒（pMMP65）构建了携带eltB的质粒。将其导入ΔlonΔcpxRΔasd鼠伤寒沙门氏菌菌株，并命名为LTB佐剂菌株。通过免疫印迹分析和酶联免疫吸附测定证实了该菌株中LTB蛋白的产生和分泌。评估了LTB菌株对先前构建的活沙门氏菌候选疫苗诱导的免疫增强和保护效果。此外，优化了使用LTB菌株的免疫策略以实现有效的沙门氏菌病保护。将70只雌性BALB/c小鼠分为7组（A至G；每组n = 10只小鼠）。小鼠在6周龄时进行初次免疫，并在9周龄时进行加强免疫。在加强免疫后第3周，所有小鼠均经口用强毒野生型菌株攻击。单独用LTB菌株免疫或用LTB菌株与候选疫苗混合物免疫的小鼠血清IgG和IgA滴度显著增加。单独用LTB菌株免疫或用混合物免疫的小鼠分泌型IgA滴度至少是对照小鼠的2.2倍。此外，所有E组小鼠（用疫苗-LTB混合物进行初次免疫并用候选疫苗进行加强免疫）均无沙门氏菌病的临床症状，并在强毒攻击中存活。相比之下，对照小鼠的攻击死亡率为100%，A组小鼠（用候选疫苗单次免疫）为80%，B组小鼠（用候选疫苗进行初次免疫和加强免疫）为60%，C组小鼠（用LTB菌株单次免疫）为40%，D组小鼠（用LTB菌株进行初次免疫和加强免疫）为30%，F组小鼠（用疫苗-LTB混合物进行初次免疫和加强免疫）为30%。这些结果表明，用LTB菌株进行疫苗接种，特别是仅在初次免疫阶段添加时，可有效增强免疫反应并预防沙门氏菌病。
