Novel multivalent vaccine for anthrax and botulinum

描述(申请人提供)：有效的疫苗增强抗体亲和力，增加T细胞寿命。使用益生菌乳杆菌(L.)表达炭疽保护性抗原(PA)融合蛋白的菌株。这种由益生菌乳杆菌表达的口服疫苗不仅能最佳地激活树突状细胞(DC)，而且还能通过来自噬菌体文库的12聚体多肽将靶向抗原(即炭疽PA)递送到粘膜DC。我们进一步优化了加瑟氏乳杆菌表达的免疫原性疫苗融合蛋白的表达。获得的数据清楚地表明，利用高拷贝载体在加瑟氏乳杆菌PA-DC融合表达中提供了强大的免疫保护，以抵御炭疽斯特恩的攻击。本研究的目的是测试这种由炭疽杆菌PA和A型肉毒梭菌神经毒素重链(HC)组成的口服多价疫苗在吸入性炭疽和BoNT/A中毒中的效果，最后检查炭疽PA-DCpep和/或HC-DCpep融合的BoNT/A C末端整合到加瑟氏弧菌染色体上的方法是否优于基于质粒的方法。适用于这项工作的三种新方法是：(A)通过使用加瑟氏乳杆菌来提高递送载体的佐剂性；(B)通过专门使用高拷贝载体进行免疫原性融合来提高疫苗效力；以及(C)通过细菌染色体插入在加瑟氏乳杆菌中表达多价免疫原性融合疫苗，避免潜在的质粒不稳定性。我们的假设是，这种优化的口服多价疫苗将在小鼠中诱导针对吸入性炭疽和BONT/A的黏膜和系统免疫反应。其具体目的是：(1)确定表达靶向炭疽PA-DCpep或BoNT/A HC-DCpep融合疫苗是否能增强对吸入性炭疽或BONT/A中毒的保护性免疫；(2)确定PA-DCpep或BoNT/A HC-DCpep融合疫苗在体内的保护效果；以及(3)确定由炭疽PA和BoNT/A-HC融合基因组成的益生菌多价疫苗对吸入性炭疽和BONT/A挑战的有效性。这一努力将通过新型佐剂来实现，这些佐剂提供疫苗的“靶向”、对致命病原体的疫苗有效性的评估，以及控制益生菌L.asseri的基因表达，这些基因表达可以很容易地被口服，从而使“靶向”抗原能够自然地递送到粘膜DC。 我们免疫系统中一组称为树突状细胞的细胞被激活，这对于正确防御不同类型的感染至关重要。这项拨款提案的目标是提供工具，利用新一代无害的免疫刺激细菌--加瑟氏乳杆菌，有效地激活粘膜和系统免疫系统，这种细菌可以作为膳食补充剂摄入。我们的数据显示，产生炭疽疫苗的加瑟氏乳杆菌对炭疽感染产生了强大的保护性免疫。因此，这一益生菌疫苗战略将被用来表明它们对于多价疫苗的交付以及刺激粘膜和系统免疫以对抗炭疽和肉毒杆菌等致命细菌的关键载体是重要的。

DESCRIPTION (provided by applicant): Effective vaccines potentiate antibody avidity and increase T cell longevity. A new generation of mucosal vaccines is being developed using probiotic Lactobacillus (L.) strains expressing anthrax-protective antigen (PA) fusion protein. Such an oral vaccine, expressed by probiotic Lactobacillus species not only optimally activates dendritic cells (DCs), but also delivers targeted antigens (i.e. anthrax PA) to mucosal DCs via 12-mer peptides derived from a bacteriophage library. We further optimized the expression of the immunogenic vaccine fusions expressed by L. gasseri. Obtained data clearly show that the use of a high copy vector for PA-DC fusion expression by L. gasseri confers robust immune protection against anthrax Sterne challenge. The objective of this proposal is to test the efficacy of such an oral multivalent vaccine consisting of anthrax PA and Clostridium botulinum neurotoxin serotype A (BoNT/A) heavy chain (Hc) in inhaled anthrax and BoNT/A intoxication and lastly to examine whether integration of the anthrax PA-DCpep and/or BoNT/A C-terminus of Hc-DCpep fusions into the L. gasseri chromosome is superior to plasmid-based approaches. Three novel approaches adapted for this work are: (a) improving adjuvanticity of the delivery vector by employing L. gasseri; (b) improving vaccine potency by specifically using a high copy vector for immunogenic fusions; and (c) expressing the multivalent immunogenic fusion vaccines in L. gasseri by bacterial chromosomal insertion and avoiding potential plasmid instability. Our hypothesis is that this optimized oral multivalent vaccine will induce mucosal and systemic immune responses against inhaled anthrax and BoNT/A in mice. The specific aims are: (1) to determine whether L. gasseri expressing targeted anthrax PA-DCpep or BoNT/A Hc-DCpep fusion vaccines enhances protective immunity against inhaled anthrax or BoNT/A intoxication; (2) to determine the protective efficacy of PA-DCpep or BoNT/A Hc-DCpep fusion vaccines in vivo, when expressed from a chromosomal location in L. gasseri; and (3) to determine the efficacy of the probiotic multivalent vaccine consisting of anthrax-PA and BoNT/A-Hc fusions against inhalational anthrax and BoNT/A challenge. This effort will be accomplished via novel adjuvants that provide "targeting" of the vaccine, evaluation of vaccine effectiveness against deadly pathogens, and the control of probiotic L. gasseri gene expression that can readily be orally consumed to enable natural delivery of "targeted" antigen to mucosal DCs. Activation of a group of cells in our immune system, called dendritic cells, is critical for proper defense against different types of infection. The goal of this grant proposal is to provide tools for efficient activation of mucosal and systemic immune system using a new generation of harmless immunostimulatory bacteria called Lactobacillus gasseri that can be ingested as dietary supplements. Our data show that L. gasseri producing anthrax vaccine optimally generated robust protective immunity against anthrax infection. Therefore, this probiotic vaccine strategy will be used to show that they are important for multivalent vaccine delivery as well as critical vehicles that stimulate mucosal and systemic immunity for combating deadly bacteria such as anthrax and botulinum.