Development of a new PDT vaccine in a single molecule

白喉-破伤风-百日咳（DTP）疫苗主要由解毒白喉毒素（DT）、破伤风毒素（TT）和百日咳毒素（PT）组成，引进该疫苗后，日本白喉-破伤风-百日咳的发病率和死亡率急剧下降。然而，在大多数发展中国家，许多儿童仍然遭受这些传染病的折磨，并因无法获得疫苗而死亡。由于接种百白破疫苗是预防这些疾病的最佳方法，而且必须是无与伦比的，因此开发一种只含有无毒素活性的免疫保护性抗原的新型百白破疫苗应该是获得理想疫苗的下一步。该项目的目的是通过将每个成分的保护区域结合在一个单一分子中，开发一种简化和定义明确的百白破疫苗。作为构建这种候选疫苗的第一步，我们构建了重组PT S1亚基- tt片段C融合蛋白。在大肠杆菌中制备了重组杂交蛋白。75-KDa融合蛋白（p75）作为可溶性分子过表达，并通过两个连续的色谱步骤纯化到接近均匀性。纯化后的p75保留了被构象表位特异性的保护性和中和性抗百日咳毒素抗体识别的能力，如抗s1单克隆抗体1B7。当给小鼠注射时，发现这种杂交蛋白是无毒的，但具有免疫原性。对破伤风有较强的保护作用，对百日咳有一定的保护作用，并能引起百日咳毒素中和抗体反应。虽然抗百日咳毒素抗体水平较低，但其中和效价与ELISA检测的抗百日咳毒素效价相关性较好，表明重组蛋白中保护表位是保守的。接下来，构建了S1-TTC和部分DT的融合蛋白，并对其进行了部分表征。SDS-PAGE和免疫印迹分析证实了这三种抗原在大肠杆菌中的表达。此外，其他抗原如HCV也被尝试与S1-TTC p75抗原融合。由这三种抗原组成的融合蛋白的研究正在进行中。少

The introduction of the diphtheria-tetanus-pertussis (DTP) vaccine which consists of mainly detoxified diphtheria toxin (DT) , tetanus toxin (TT) , and pertussis toxin (PT) led to a dramatic decline in incidence and mortality of DTP in Japan. However, many children in the most of developing countries are still suffered with these infectious diseases and dying because of unavailability of the vaccine. Since the vaccination with DTP is the best way and must be peerless to prevent these diseases, development of a new DTP vaccine which include only immunoprotective antigens with no toxin activity should be next step towards obtaining an ideal vaccines.The purpose of this project is development of a simplified and well-defined DTP vaccine by combining the protective regions of each component in a single molecule. As a first step towards such a vaccine candidate, a recombinant PT S1 subunit-TT fragment C fusion protein was constructed. The recombinant hybrid protein was produced in Escherich … More ia coli. The 75-KDa fusion protein (p75) was over expressed as a soluble molecule and purified to near homogeneity by two consecutive chromatographic steps. Purified p75 retained its ability to be recognized by protective and neutralizing anti-pertussis toxin antibodies specific for conformational epitopes, such as anti-S1 mono-clonal antibody 1B7. When administered to mice, the hybrid protein was found to be nontoxic but immunogenic. It was capable of inducing strong protection against tetanus and some protection against pertussis, as well as eliciting a pertussis toxin-neutralizing antibody response. Although the levels of anti-pertussis toxin antibodies were rather low, their neutralizing titers correlated well with anti-pertussis toxin titers by ELISA,indicating that protective epitopes are conserved in the recombinant protein.Next, fusion proteins of S1-TTC and a part of DT,were constructed and characterized partially. Expression of these three antigens in E.coli was recognized by the SDS-PAGE and immunoblotting analysis. Furthermore, additional antigens such as HCV were attempted to fuse to the S1-TTC p75 antigen. Studies on the fusion proteins consisting of these triple antigens are in progress. Less