Development of a new PDT vaccine in a single molecule

主要由解毒白喉毒素(DT)、破伤风毒素(TT)和百日咳毒素(PT)组成的白喉-破伤风-百日咳(DTP)疫苗的引入，使日本DTP的发病率和死亡率显著下降。然而，大多数发展中国家的许多儿童仍然患有这些传染病，并因得不到疫苗而死亡。由于接种DTP疫苗是预防这些疾病的最好方法，而且必须是无与伦比的，因此开发一种新的DTP疫苗是获得理想疫苗的下一步，该疫苗只包含没有毒素活性的免疫保护性抗原。本项目的目的是通过在单个分子中结合各组分的保护区来开发一种简单且定义明确的DTP疫苗。作为疫苗候选的第一步，构建了重组PTS1亚单位-TT片段C融合蛋白。重组杂合蛋白在Escherich…中表达。更多的大肠埃希氏菌。P75融合蛋白以可溶性分子形式高效表达，连续两步层析纯化至接近均一的水平。纯化的p75仍能被针对构象表位的保护性和中和性抗百日咳毒素抗体识别，如抗S1单抗1B7。当给小鼠注射时，这种杂交蛋白被发现是无毒的，但具有免疫原性。它能诱导对破伤风的强大保护和对百日咳的一些保护，并能引起百日咳毒素中和抗体反应。虽然重组蛋白中抗百日咳毒素抗体水平较低，但其中和效价与抗百日咳毒素效价有很好的相关性，表明重组蛋白中含有保护性表位。经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，还尝试了其他抗原，如丙型肝炎病毒，以与S1-TTC p75抗原融合。对由这三种抗原组成的融合蛋白的研究正在进行中。较少

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