Immunogenicity of an Attenuated Listeria monocytogenes Bacteriophage Resistant Mu

描述(由申请人提供)：本R21申请的目的是研究噬菌体抗药性如何在保持单核细胞增多性李斯特菌对小鼠口服毒力的免疫原性的同时提供减毒作用。李斯特菌菌株F6214-1对噬菌体P35h4的抗性突变株在口服接种雌性A/J小鼠时会减弱，并在培养的小鼠肠道细胞中表现出复制障碍。其中一个突变体在glcV基因中包含Tn917插入，已被广泛鉴定。噬菌体结合研究表明，突变体的细胞表面发生了改变，排除了噬菌体的附着。与突变相关的所有表型在反式中由glcV基因的双亲拷贝补充。据预测，glcV基因编码2组糖基转移酶。该产物的丢失会导致噬菌体受体的缺陷，并改变毒力所需的正常宿主-病原体相互作用。有趣的是，glcv损伤虽然阻止了噬菌体附着，但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，即斑块形成效率和斑块大小的降低。在体内，与高侵袭性亲本菌株相比，在口服接种48h后，在肝和脾中检测不到突变体。该突变体很重要，因为它的记录特性(和我们的初步研究)表明了它作为口服活疫苗平台的突出潜力。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变化的性质、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阶段的不完全了解。因此，我们的具体目标是针对(1)消除噬菌体结合的细菌细胞表面缺陷的性质，(2)体外抗噬菌体突变体的生长和细胞间传播的缺陷，以及(3)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，我们的研究结果将对我们对自然获得性李斯特菌病(即通过口服接种途径获得的)发病机制的步骤的理解有实质性的改进。此外，这些特性可能支持噬菌体抗性突变体作为疫苗平台的进一步发展。这样的平台有可能提供一种实用、安全和有效的方式，通过这种方式将抗原呈递到免疫系统。这可能导致预防传染病的疫苗和对抗肿瘤生长的免疫疗法的改进。公共卫生相关性：我们将研究噬菌体抗药性如何在保持小鼠口服毒力的单核细胞增多性李斯特菌菌株的免疫原性的同时提供减毒作用。我们认为，我们的研究结果将对我们对自然形式李斯特菌病发病机制的步骤(即通过口服接种途径)的理解有实质性的改善。此外，这些特征可能支持抗噬菌体突变体作为疫苗平台的发展。这样的平台有可能提供一种实用、安全和有效的方法，将抗原呈递到免疫系统，导致预防传染病的疫苗和对抗肿瘤生长的免疫疗法的改进。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