Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection
ZHANG Jing, CUI Ya-Li, JIANG Yong-Mei
Department of Clinical Laboratory, West China Second University Hospital, Sichuan University/Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, Chengdu 610041, China
摘要 目的 原核表达肺炎链球菌内肽酶O(PepO)及肺炎链球菌表面黏附素A(PsaA),评价其作为疫苗候选蛋白的免疫保护效果。方法 设计pepO,psaA目的基因片段的特异性引物,经PCR扩增,构建重组质粒pET28a(+)-pepO,pET28a(+)-psaA,重组质粒转化大肠埃希菌BL21(DE3)诱导表达,经纯化后获得高纯度目的蛋白PepO及PsaA。经黏膜免疫BALB/c小鼠制备其特异性抗血清,ELISA检测抗体效价,Western blot分析验证目的蛋白抗血清的特异性。将BALB/c小鼠随机分为阴性对照组、PepO组、PsaA组及PepO+PsaA联合免疫组,每组18只。构建不同血清型肺炎链球菌感染模型,评估目的蛋白单用及联合使用的免疫保护效果。结果 成功制备并获得目的蛋白PepO及PsaA,经Western blot验证目的蛋白抗血清特异性好。PepO组和联合免疫组小鼠唾液中IgA及血清中IgG效价比较差异无统计学意义(P > 0.05),但均高于PsaA组(P P P P P 结论 PepO+PsaA联合疫苗经黏膜途径免疫小鼠后,较单用对小鼠有更好的保护作用,能有效抵抗肺炎链球菌在鼻咽部、肺部的定植,是一组较有开发潜力的蛋白疫苗。
Abstract:Objective To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins. Methods Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a (+)-pepO and pET28a (+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination. Results The target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P > 0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P Streptococcus pneumoniae D39 than the PsaA group (P Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P Conclusions Combined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.
ZHANG Jing,CUI Ya-Li,JIANG Yong-Mei. Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection[J]. CJCP, 2017, 19(5): 583-589.
World Health Organization. Estimates of disease burden and cost effectiveness[EB/OL]. (2016-11-11)[2016-11-22].
[2]
O'Brien KL, Wolfson LJ, Watt JP, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years:global estimates[J]. Lancet, 2009, 374(9693):893-902.
[3]
Braido F, Bellotti M, De Maria A, et al. The role of Pneumococcal vaccine[J]. Pulm Pharmacol Ther, 2008, 21(4):608-615.
[4]
Kristian SA, Ota T, Bubeck SS, et al. Generation and improvement of effector function of a novel broadly reactive and protective monoclonal antibody against pneumococcal surface protein A of Streptococcus pneumoniae[J]. PLoS One, 2016, 11(5):e0154616.
[5]
Balachandran P, Brooks-Walter A, Virolainen-Julkunen A, et al. Role of pneumococcal surface protein C in nasopharyngeal carriage and pneumonia and its ability to elicit protection against carriage of Streptococcus pneumoniae[J]. Infect Immun, 2002, 70(5):2526-2534.
[6]
Tai SS. Streptococcus pneumoniae protein vaccine candidates:properties, activities and animal studies[J]. Crit Rev Microbiol, 2006, 32(3):139-153.
[7]
Salha D, Szeto J, Myers L, et al. Neutralizing antibodies elicited by a novel detoxified pneumolysin derivative, PlyD1, provide protection against both pneumococcal infection and lung injury[J]. Infect Immun, 2012, 80(6):2212-2220.
[8]
Manco S, Hernon F, Yesilkaya H, et al. Pneumococcal neuraminidases A and B both have essential roles during infection of the respiratory tract and sepsis[J]. Infect Immun, 2006, 74(7):4014-4020.
[9]
Shaik MM, Maccagni A, Tourcier G, et al. Structural basis of pilus anchoring by the ancillary pilin RrgC of Streptococcus pneumoniae[J]. J Biol Chem, 2014, 289(24):16988-16997.
[10]
Harfouche C, Filippini S, Gianfaldoni C, et al. RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies[J]. Infect Immun, 2012, 80(1):451-460.
[11]
Cui Y, Zhang X, Gong Y, et al. Immunization with DnaJ (hsp40) could elicit protection against nasopharyngeal colonization and invasive infection caused by different strains of Streptococcus pneumonia[J]. Vaccine, 2011, 29(9):1736-1744.
[12]
Liu Y, Wang H, Zhang S, et al. Mucosal immunization with recombinant fusion protein DnaJ-ΔA146Ply enhances cross-protective immunity against Streptococcus pneumoniae infection in mice via interleukin 17A[J]. Infect Immun, 2014, 82(4):1666-1675.
[13]
Agarwal V, Kuchipudi A, Fulde M, et al. Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional plasminogen-and fibronectin-binding protein, facilitating evasion of innate immunity and invasion of host cells[J]. J Biol Chem, 2013, 288(10):6849-6863.
[14]
Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectin extracellular matrix[J]. Annu Rev Cell Dev Biol, 2010, 26:397-419.
[15]
Schwarz-Linek U, Höök M, Potts JR. The molecular basis of fibronectin-mediated bacterial adherence to host cells[J]. Mol Microbiol, 2004, 52(3):631-641.
[16]
Collen D, Verstraete M. Molecular biology of human plasminogen. II. Metabolism in physiological and some pathological conditions in man[J]. Thromb Diath Haemorrh, 1975, 34(2):403-408.
[17]
Novak R, Braun JS, Charpentier E, et al. Penicillin tolerance genes of Streptococcus pneumoniae:the ABC-type manganese permease complex Psa[J]. Mol Microbiol, 1998, 29(5):1285-1296.
[18]
Johnston JW, Myers LE, Ochs MM, et al. Lipoprotein PsaA in virulence of Streptococcus pneumoniae:surface accessibility and role in protection from superoxide[J]. Infect Immun, 2004, 72(10):5858-5867.
[19]
McAllister LJ, Tseng HJ, Ogunniyi AD, et al. Molecular analysis of the psa permease complex of Streptococcus pneumoniae[J]. Mol Microbiol, 2004, 53(3):889-901.
[20]
Singh R, Gupta P, Sharma PK, et al. Prediction and characterization of helper T-cell epitopes from pneumococcal surface adhesin A[J]. Immunology, 2014, 141(4):514-530.
[21]
De Magistris MT. Mucosal delivery of vaccine antigens and its advantages in pediatrics[J]. Adv Drug Deliv Rev, 2006, 58(1):52-67.
[22]
Ferreira DM, Darrieux M, Silva DA, et al. Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice[J]. Clin Vaccine Immunol, 2009, 16(5):636-645.
[23]
Zygmunt BM, Rharbaoui F, Groebe L, et al. Intranasal immunization promotes Th17 immune responses[J]. J Immunol, 2009, 183(11):6933-6938.
[24]
Neutra MR, Kozlowski PA. Mucosal vaccines:the promise and the challenge[J]. Nat Rev Immunol, 2006, 6(2):148-158.
[25]
Liu Y, Wang H, Chen M, et al. Serotype distribution and antimicrobial resistance patterns of Streptococcus pneumoniae isolated from children in China younger than 5 years[J]. Diagn Microbiol Infect Dis, 2008, 61(3):256-263.