Huang Xiaohui, Li Yu, Fu Yuguang, Ji Yanhong, Lian Kaiqi, Zheng Haixue, Wei Jianzhong, Cai Xuepeng, Zhu Qiyun
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, People's Republic of China.
Clin Vaccine Immunol. 2013 Jun;20(6):912-9. doi: 10.1128/CVI.00621-12. Epub 2013 Apr 24.
The causative agent of Glasser's disease in swine is Haemophilus parasuis. Commercial bacterins are widely used for protection of the swine population. However, cross protection is limited because H. parasuis has more than 15 serovars. Transferrin-binding protein A has shown potential as a broad-spectrum vaccine candidate against homologous and heterologous strains. Here we amplified the full-length tbpA gene from an H. parasuis serovar 13 isolate and cloned it into a pET-SUMO expression vector. We then expressed and purified the TbpA protein by Ni affinity chromatography. First, the immunogenicity and protective efficacy of the protein were evaluated in guinea pigs by two subcutaneous immunizations with different doses of Montanide IMS 206 VG adjuvant. The immunized guinea pigs were, respectively, challenged on week 3 after a booster immunization with homologous strain LJ3 (serovar 13) and heterologous strain FX1 (serovar 4), and vaccine-inoculated groups were compared with nonvaccinated controls. All immunized groups showed serum antibody titers higher than those of negative-control groups. Furthermore, the cytokine and chemokine levels were evaluated at the transcriptional level by the real-time PCR analysis of six cytokines and chemokines. Gamma interferon and interleukin-5 in groups immunized with 100 μg were elevated more than 15-fold over those in negative-control groups. The protection rates were 80 and 60% after a challenge with strains LJ3 and FX1, respectively, in the groups vaccinated with 100 μg of recombinant TbpA protein. Subsequently, the data showed that guinea pigs immunized with a single dose (100 μg) were protected at levels of 80, 80, and 60% against LJ3, FX1, and another heterologous strain, SZ (serovar 14), respectively. The results indicate for the first time that TbpA protein cross protects guinea pigs against serovars 13, 4, and 14 of H. parasuis. Taken together, these results suggest that the recombinant TbpA protein is a promising vaccine candidate that needs to be confirmed in a swine population.
猪格拉泽氏病的病原体是副猪嗜血杆菌。商业菌苗被广泛用于保护猪群。然而，由于副猪嗜血杆菌有超过15个血清型，交叉保护作用有限。转铁蛋白结合蛋白A已显示出作为针对同源和异源菌株的广谱疫苗候选物的潜力。在此，我们从一株副猪嗜血杆菌血清型13分离株中扩增出全长tbpA基因，并将其克隆到pET-SUMO表达载体中。然后，我们通过镍亲和层析表达并纯化了TbpA蛋白。首先，用不同剂量的Montanide IMS 206 VG佐剂进行两次皮下免疫，在豚鼠中评估该蛋白的免疫原性和保护效力。在加强免疫后第3周，分别用同源菌株LJ3（血清型13）和异源菌株FX1（血清型4）对免疫的豚鼠进行攻毒，并将接种疫苗的组与未接种疫苗的对照组进行比较。所有免疫组的血清抗体滴度均高于阴性对照组。此外，通过对六种细胞因子和趋化因子进行实时PCR分析，在转录水平评估细胞因子和趋化因子水平。用100μg免疫的组中，γ干扰素和白细胞介素-5比阴性对照组升高了15倍以上。在用100μg重组TbpA蛋白接种的组中，用LJ3和FX1菌株攻毒后的保护率分别为80%和60%。随后，数据显示，用单剂量（100μg）免疫的豚鼠分别对LJ3、FX1和另一种异源菌株SZ（血清型14）的保护率为80%、80%和60%。结果首次表明，TbpA蛋白可交叉保护豚鼠抵抗副猪嗜血杆菌的血清型13、4和14。综上所述，这些结果表明重组TbpA蛋白是一种有前景的疫苗候选物，需要在猪群中进行验证。
