肺炎克雷伯菌耐药靶标KPC-2的免疫原性和保护效果评价及保护机制初步研究

Immunogenicity, protective efficacy and preliminary protective mechanism of KPC-2, a drug resistance target from Klebsiella pneumoniae

目的采用肺炎克雷伯菌耐药靶标KPC-2为抗原制备重组蛋白疫苗,在小鼠肺炎模型中评价疫苗的免疫原性、保护效果和保护机制。方法利用大肠埃希菌原核表达系统表达肺炎克雷伯菌KPC-2蛋白,经GST亲和层析对目的蛋白进行纯化;采用KPC-2蛋白制备疫苗,经皮下注射免疫新西兰兔,心脏采血分离血清,用Protein G亲和层析纯化多克隆抗体,同时运用调理吞噬杀菌试验检测抗体的体外杀菌活性。采用KPC-2疫苗免疫雌性BALB/c小鼠3次,间接ELISA检测血清中特异性IgG抗体滴度。末次免疫后7 d,通过气管插管构建小鼠肺炎克雷伯菌肺部感染模型,感染1 h后尾静脉给予0.1 mg美罗培南治疗,通过比较免疫组与佐剂组的生存率、细菌定植量和组织病理学差异,以及给药组与生理盐水组生存率的差异评价疫苗的保护效果。采用KPC-2多克隆抗体被动免疫评价抗体的保护效果。结果KPC-2免疫组小鼠血清特异性IgG水平显著高于对照组(t=4.325,P<0.05),免疫组生存率显著高于对照组[70%(7/10)vs 10%(1/10),P<0.05];免疫组小鼠肺组织炎性程度及细菌定植量显著低于对照组(t=3.127,P<0.05);疫苗主动免疫与被动免疫均具有良好的保护效果,且对抗生素治疗有明显的增效性;KPC-2多克隆抗体在体外表现出明显的吞噬杀菌活性(t=5.427,P<0.05)。结论KPC-2疫苗具有良好的免疫原性和保护效果,体液免疫应答在其保护中发挥了重要作用,证明采用耐药靶标作为抗原制备疫苗具有可行性。

Objective To develop a recombinant protein vaccine based on KPC-2,a drug resistance target in Klebsiella pneumoniae,and evaluate its immunogenicity,protective efficacy and mechanism in a mouse model of pneumonia.Methods KPC-2 was expressed in Escherichia coli and purified using GST affinity chromatography.A recombinant protein vaccine was prepared with KPC-2 and used to immunize New Zealand rabbits through subcutaneous injection.Serum samples were isolated from cardiac blood and Protein G chromatography was used to purify polyclonal antibodies against KPC-2.Opsonophagocytic killing assay was used to assess the bactericidal activity of the polyclonal antibodies in vitro.Female BALB/c mice were immunized three times with the recombinant protein vaccine,and the titers of specific IgG antibodies in serum were measured by indirect ELISA.One week after the last vaccination,the mice were infected with Klebsiella pneumoniae strain SRT through tracheal intubation,and received a single intravenous dose of meropenem(0.1 mg)1 h later.The protective efficacy of the KPC-2 recombinant protein vaccine was evaluated by comparing the survival rates,bacterial colonization and histopathological changes between vaccine group and adjuvant group as well as the survival rates between meropenem group and normal saline group.Moreover,the protective efficacy of polyclonal antibodies against KPC-2 was evaluated through passive immunization.Results The level of specific IgG antibodies in serum was significantly higher in the vaccine group than in the adjuvant group(t=4.325,P<0.05).The survival rate in the vaccine group was also higher than that of the adjuvant group[70%(7/10)vs 10%(1/10),P<0.05].Furthermore,lung inflammation was less severe and bacterial burden was reduced in the vaccine group as compared with those of the control group(t=3.127,P<0.05).Both active and passive vaccination strategies demonstrated strong protective efficacy against Klebsiella pneumoniae infection,and had a synergistic effect when used in combination with antibiotic therapy.The polyclonal antibodies against KPC-2 had bactericidal activity in vitro(t=5.427,P<0.05).Conclusions The prepared KPC-2 vaccine has better immunogenicity and protective efficacy.It can induce strong humoral immune responses.This study suggest that drug resistance target may be used as a candidate antigen for future vaccine development.