纳米细菌纤维素制备含有细菌佐剂并具有体内DC疫苗特性的疟疾疫苗

Dendritic cell (DC) vaccine is a good approach for inducing cell immune response, but it needs a series complicated handling-procedures in vitro, leading to difficulty in technical standardization. MIP-3α is a special chemokine of DC. Recently, we had made a special patent microparticle using a nano-medical material, bacterial cellulose. Our previous study indicated that the bacterial-cellulose microparticles encapsulated with tumor cells can keep the tumor cell alive for several days and the live tumor cells can also keep secreting cellular active molecules. Such bacterial-cellulose microparticles have the capabilities of inducing strong immune response against tumor cells when injected into mouse as vaccine. As we know, bacterial cellulose is a material that completely different from plant cellulose (such as cotton), bacterial cellulose possess excellent abilities, such as histocompatibility, biodegradability, and permeability, leading to its potential widespread usage as medical materials. In this study, we will translate this idea to develop a novel approach for developing a malaria vaccine. We will apply transgenic technique to construct a MIP-3α transgenic malaria merozoite of blood stage. We will then make an injectable microparticle using our patent nano-bacterial cellulose as the major materials, in which MIP-3α transgenic malaria merozoites and inactivated Salmonella are encapsulated. We thus name these special microparticles as "merozoite bacterial microparticales". These "merozoite bacterial microparticles" possess many new characteristics, such as secretion of DC chemokine MIP-3α by the transgenic merozoite, slow release of the merozoite (we try to keep it alive and secretes MIP-3α only several days) and Salmonella antigens, which will promote DC and other immunocytes to reach the microparticle-injected side and process the antigens of both merozoites and Salmonella, leading to strong immune response against malaria merozoites. In addition, we will also use the "merozoite bacterial microparticale" as a malaria vaccine to investigate its anti-malaria activities in murine malaria models. Moreover, modern biomedical techniques, such as immunology and molecular biology, will be used to further unveil the possible mechanisms by which the "merozoite bacterial microparticale" vaccine works. Thus, it may be possible that our novel "merozoite bacterial microparticale" as a vaccine has the possibility of becoming the simplest and most effective approach for inducing immune response against malaria. Because our proposal applies the "area－subjects" which only support the researchers from remote and economically underdeveloped areas in china. To our knowledge, we think our present proposal belong to the good or better category amongst the area subjects. Thus, we think it is worth for reviewers to support our proposal.

DC疫苗是诱导细胞免疫的好方法，但需要在体外进行复杂的技术操作，技术标准化难。MIP-3α是DC 特异性趋化因子。本项目利用转基因技术将MIP-3α转进红内期疟原虫，同时应用专利技术制备可注射的细菌纤维素颗粒，并将转基因疟原虫感染的红细胞和灭活沙门氏菌共包埋于颗粒中。这种"颗粒"中的疟原虫可以分泌MIP-3α并且只能存活一段时间。借助细菌纤维素生物相容、可降解和对分子通透的特性，让MIP-3α、死亡的疟原虫和细菌抗原缓慢释放，吸引DC和其它多种免疫（炎症）细胞，在注射部位造成一个适合免疫反应的微环境，最终免疫细胞将完全吸收细菌纤维素颗粒，从而诱导并增强抗疟原虫的免疫反应。这种"细菌纤维素颗粒"有可能是一种简单而有效的体内疟疾DC疫苗。同时，建立体内外疟疾模型，用"细菌纤维素颗粒"作为疫苗，了解是否有防治疟疾的作用，用免疫学、分子生物学等技术证明这种疫苗的作用机理，寻求一种疟疾防治的新方法。

尽管有青蒿素等特效疟疾防治药物，但疟疾耐药也正在漫延，由于疟疾目前主要在发展中国家，尤其是在非洲的不发达因流行较广，投入新药开发和疫苗研究的资金十分有限，加上还没有真正有效的防治疫苗，因此开发新型疟疾防治疫苗还是一项重要的研究课题。DC细胞是最强有力的抗原递呈细胞，在体内招募聚集DCs病原感染部位（或直接作用于感染细胞，如本研究的疟原虫感染的红细胞）是一种有效的体内DC疫苗方式。当前，用肿瘤细胞裂解物与自体DC细胞在体外混合刺激的DC疫苗、巨噬细胞炎症蛋白3α（MIP-3α）的表达载体注入肿瘤部位的体内DC疫苗、利用各种办法逆转肿瘤组织中的免疫抑制微环境等免疫生物治疗肿瘤的办法已经有文献报道，但是尚没有类似疟疾疫苗开发的报道。因此，将体内DC疫苗等多种研究策略（思路）综合于一体并用于制备针对疟疾感染红细胞的疫苗便是一种新的技术方法。本项目利用转基因工程技术将DC趋化因子MIP-3α基因转入小鼠疟原虫——伯氏疟原虫，在体内外试验表明可以这一种疟原虫能有效表达活性功能的MIP-3α（其中包括体外试验具有趋化吸引DCs的功能）后，将这一种伯氏疟原虫感染的红细胞提取出来，然后用细菌纤维素将感染的红细胞和灭活的沙门氏细菌包被成一种可注射的微颗粒（称为MIP-3α细菌纤维素颗粒或PBcMIP3α）。体外检测发现MIP-3α细菌纤维素颗粒可以有效表达并分泌具有功能活性的MIP3α蛋白，并且在体外和注射的腹腔（体内）都发现具有明显的趋化DC的作用；此后将MIP-3α细菌纤维素颗粒作为疫苗，在小鼠伯氏疟原虫模型中发现具有明显的防治疟疾的作用，能够有效诱导产生感染红细胞的免疫反应。进一步进行机理研究发现，脾脏中发现MIP-3α细菌纤维素颗粒免疫有更多分泌干扰素-γ的CD8和CD4淋巴细胞；用ELISA和Western Blot、ELISPOT等方法检测体液免疫也发现，MIP-3α细菌纤维素颗粒免疫也可以诱导疟原虫感染红细胞特异的抗体，主要抗体成分为IgG1和IgG2a。用多种技术检测发现，MIP-3α细菌纤维素颗粒疫苗能够覆盖感染红细胞表面的CD47，从而让增加巨噬细胞和DCs对其吞噬和抗原处理的能力。这些结果表明，MIP-3α细菌纤维素颗粒是一种有效疟疾疫苗制备技术，具有一定转化应用前景。