Immunogenicity of an Attenuated Listeria monocytogenes Bacteriophage Resistant Mu

描述（由申请人提供）：本R21申请的目的是检查噬菌体抗性如何赋予减毒，同时保留对小鼠具有口服毒性的单核细胞增生李斯特菌菌株的免疫原性。对噬菌体P35 h4具有抗性的Escherial菌株F6214-1的突变体在经口接种到雌性A/J小鼠中时减弱，并且在培养的小鼠肠上皮细胞中显示出复制受损。这些突变体之一，包含一个Tn 917插入在glcV基因，已被广泛的特点。噬菌体结合研究表明，突变体具有细胞表面改变，排除噬菌体附着。与突变相关的所有表型都由glcV基因的亲本拷贝反式补充。预测glcV基因编码第2组糖基转移酶。这种产物的丢失导致噬菌体受体缺陷，并改变了毒力所需的正常宿主-病原体相互作用。有趣的是，glcV病变在阻止噬菌体附着的同时，并不影响突变体与培养的小鼠肠细胞单层结合的能力。相反，突变似乎改变了细胞内复制的后续步骤，测量为斑块形成效率和斑块大小的降低。在体内，突变体是，在相反的高度侵入性的亲本菌株，在肝脏和脾脏中检测不到口服接种后48小时。该突变体很重要，因为其记录的特性（以及我们的初步研究）表明其作为活口服疫苗平台的出色潜力。以这种方式利用突变体的障碍包括对排除噬菌体结合的细胞表面改变的性质、突变体的细胞内生长特性以及宿主根除突变体的感染过程中的阶段的不完全理解。因此，我们的具体目标是针对以下的表征：（1）消除噬菌体结合的细菌细胞表面缺陷的性质，（2）体外噬菌体抗性突变体的生长和细胞间扩散的缺陷，和（3）在突变体从肠腔移位后器官参与和免疫发展的程度。我们认为，我们的研究结果将构成我们对自然获得性乳腺癌发病机制步骤的理解的实质性改进（即，通过口服接种途径获得）。此外，表征可以支持噬菌体抗性突变体作为疫苗平台的进一步开发。这种平台有可能提供一种实用、安全和有效的手段，通过这种手段将抗原呈递给免疫系统。这可能导致预防传染病的疫苗和对抗肿瘤生长的免疫治疗的改进。公共卫生相关性：我们将研究如何噬菌体抗性赋予衰减，同时保留免疫原性的小鼠口服毒力单核细胞增生李斯特菌菌株。我们认为，我们的研究结果将构成我们对自然形式的阿尔茨海默病的发病机制步骤的理解的实质性改进（即，通过口服接种途径）。此外，所述表征可以支持噬菌体抗性突变体作为疫苗平台的开发。这种平台有可能提供一种实用、安全和有效的手段，通过这种手段将抗原呈递给免疫系统，从而改善预防传染病的疫苗和对抗肿瘤生长的免疫治疗。2

DESCRIPTION (provided by applicant): The purpose of this R21 application is to examine how bacteriophage resistance confers attenuation, while preserving immunogenicity of a Listeria monocytogenes strain that is oral-virulent for mice. Mutants of listerial strain F6214-1 that are resistant to phage P35h4 are attenuated when inoculated orally into female A/J mice and show impaired replication in cultured mouse enterocytes. One of these mutants, containing a Tn917 insertion in the glcV gene, has been extensively characterized. Phage binding studies indicate that the mutant has a cell surface alteration that precludes phage attachment. All phenotypes associated with the mutation are complemented in trans by a parental copy of the glcV gene. The glcV gene is predicted to encode a group 2 glycosyl transferase. The loss of this product results in a defective phage receptor and alters a normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, does not affect the mutant's ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appears to alter a subsequent step in intracellular replication measured as a reduction in plaque forming efficiency and plaque size. In vivo, the mutant is, in contrast to the highly invasive parent strain, undetectable in the liver and spleen 48 h post oral inoculation. The mutant is important because its documented properties (and our preliminary studies) indicate its outstanding potential as a live oral vaccine platform. Impediments to exploiting the mutant in this fashion include an incomplete understanding of the nature of the cell surface alteration that precludes phage binding, the mutant's intracellular growth properties, and the stage in the infectious process when the host eradicates the mutant. Accordingly, our specific aims are directed towards a characterization of (1) the nature of the bacterial cell surface defect that eliminates phage binding, (2) the defect in growth and cell-to-cell spread of the phage resistant mutant in vitro, and (3) the extent of organ involvement and development of immunity following the translocation of the mutant from the intestinal lumen. We think that the results of our studies will constitute a substantive improvement in our understanding of the steps in the pathogenesis of naturally acquired listeriosis (i.e., acquired via an oral inoculation route). Furthermore, the characterizations may support the further development of the phage resistant mutant as a vaccine platform. Such a platform has the potential to provide a practical, safe and efficient means by which antigens are presented to the immune system. This could lead to improvements in vaccines to prevent infectious disease and in immunotherapeutics to combat tumor growth. PUBLIC HEALTH RELEVANCE: We will examine how phage resistance confers attenuation while preserving immunogenicity in a mouse oral-virulent Listeria monocytogenes strain. We feel that the results of our studies will constitute a substantive improvement in our understanding of the steps in the pathogenesis of the natural form of listeriosis (i.e., via an oral inoculation route). Further, the characterizations may support the development of the phage resistant mutant as a vaccine platform. Such a platform has the potential to provide a practical, safe, and efficient means by which antigens are presented to the immune system, leading to improvements in vaccines to prevent infectious disease and in immunotherapeutics to combat tumor growth. 2