Kazemian Hamid, Afshar Davoud, Garcia Ernesto, Pourmand Mohammad Reza, Jeddi-Tehrani Mahmood, Aminharati Farzaneh, Shokri Fazel, Yazdi Mohammad Hossein
Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Madrid, Spain.
Iran J Allergy Asthma Immunol. 2018 Dec 3;17(6):574-585.
Among many pneumococcal antigens, choline-binding proteins (CPBs) display a high immunogenicity in animal models. This study aims to determine the immunogenicity of CbpM, CbpG and CbpL proteins of Streptococcus pneumoniae in a mice model. The genes were cloned into pET21a expression vector and the recombinant proteins were produced. Mice were immunized with the purified recombinant proteins. Subsequently, the mice were challenged with S. pneumoniae ATCC 49619 (2×106 CFU) and their survival and bacterial clearances were followed 24 hours after infection. The antibody responses of the mice were determined by ELISA assay. The opsonophagocytosis assay was performed using rabbit's sera. Passive immunization was carried out using two doses of anti-CbPs antibodies. Finally, these mice were experimentally infected with virulent bacteria and the protective effects of two doses of 10 and 100 µg/mL by monitoring the survival rate and bacterial clearance were determined at 2, 3 and 7 days after bacterial challenge. The mice actively immunized with CbpM, CbpG and CbpL recombinant proteins showed survival rate of 100%, 85% and 75%, respectively. The survival rates among passively immunized mice groups which received 100 µg/mL dose of anti-CbpM, anti-CbpG and anti- CbpL were 50%, 50% and 25%, respectively. The rates of opsonization with rabbit's antibodies against CbpM, CbpG, and CbpL at 100 µg/mL doses was 45.6%, 14.7% and 82.3%, and at 10 µg/mL was 12.9%, 12.2% and 9.35%, respectively. Our findings suggest that the recombinant proteins particularly CbpM and CbpG can protect the mice against pneumococcus19F serotype and effectively induce a protective antibody response. Thus, CbpG and CbpM proteins might be used as suitable vaccine candidate in pneumococcal vaccine formulations.
在众多肺炎球菌抗原中，胆碱结合蛋白（CPBs）在动物模型中表现出高免疫原性。本研究旨在确定肺炎链球菌的CbpM、CbpG和CbpL蛋白在小鼠模型中的免疫原性。将这些基因克隆到pET21a表达载体中并产生重组蛋白。用纯化的重组蛋白免疫小鼠。随后，用肺炎链球菌ATCC 49619（2×10⁶ CFU）攻击小鼠，并在感染后24小时观察其存活率和细菌清除情况。通过ELISA测定法确定小鼠的抗体反应。使用兔血清进行调理吞噬作用测定。使用两剂抗CPBs抗体进行被动免疫。最后，用有毒力的细菌对这些小鼠进行实验性感染，并通过监测存活率和细菌清除情况，在细菌攻击后2、3和7天确定两剂10和100 µg/mL的保护作用。用CbpM、CbpG和CbpL重组蛋白主动免疫的小鼠存活率分别为100%、85%和75%。接受100 µg/mL剂量抗CbpM、抗CbpG和抗CbpL的被动免疫小鼠组的存活率分别为50%、50%和25%。100 µg/mL剂量的兔抗CbpM、CbpG和CbpL抗体的调理作用率分别为45.6%、14.7%和82.3%，10 µg/mL时分别为12.9%、12.2%和9.35%。我们的研究结果表明，重组蛋白尤其是CbpM和CbpG可以保护小鼠免受19F血清型肺炎球菌的侵害，并有效诱导保护性抗体反应。因此，CbpG和CbpM蛋白可能用作肺炎球菌疫苗制剂中合适的疫苗候选物。
