EPITOPE DELIVERY, IMMUNODOMINANCE AND VACCINE EFFICACY

描述（改编自申请人摘要）：对单核增生乳杆菌的保护性免疫反应用于研究细胞介导的细胞内细菌免疫的一般特征。在BALB/c小鼠中，分离到了Kd限制的李斯特菌特异性CD8+ CTL。从吞噬液泡中分泌的利斯特溶素O （LLO）分子和细胞质中大量分泌的p60分子中提取的肽已被确定为CTL的靶点。CTL频率分析显示，LLO衍生的决定因子LLO 91-99具有免疫优势。我们推测，吞噬液泡内LLO的分泌是导致LLO 91-99肽免疫优势的一个因素。在Specific Aim I中，研究将确定吞噬液泡内特定肽的分泌是否是其免疫优势的促成因素。在这些研究中，我们将使用单核增生乳杆菌突变体，其中kd结合纳米肽（通常是酪氨酸）2位的锚定残基已被苯丙氨酸取代。突变体保留了野生型表型，但在用这些菌株免疫后，特异性CTL不会被刺激到突变肽序列。我们将使用这些突变体将特定的李斯特菌衍生肽递送到单核增生乳杆菌感染细胞的吞噬液泡或细胞质中，以确定这些初始位置对随后CTL反应的影响。单核增生乳杆菌被认为是一种传递候选抗原和刺激特异性细胞介导免疫的载体。用于递送候选抗原的疫苗载体的价值部分取决于在现有抗媒介免疫环境中特异性刺激免疫反应的能力。在特异性目标II中，我们将确定单核增生乳杆菌作为疫苗载体的效用，并评估对疫苗载体具有确定现有免疫水平的动物的肽特异性免疫的发展。我们将评估肽特异性效应细胞和记忆细胞反应的发展。这些研究的结果将指导新疫苗载体的总体开发战略，并确定其在对疫苗载体免疫状况未知的人群中的使用限制。

DESCRIPTION (adapted from applicant's abstract): The protective immune response to L. monocytogenes is used to investigate general features of cell-mediated immunity to intracellular bacteria. In BALB/c mice, Listeria-specific CD8+ CTL have been isolated which are Kd restricted. Peptides from listeriolysin O (LLO) a molecule secreted in the phagocytic vacuole, and p60, a molecule that is abundantly secreted in the cytoplasm, have been identified as CTL targets. CTL frequency analysis shows that the LLO-derived determinant, LLO 91-99, is immunodominant. We hypothesize that secretion of LLO within the phagocytic vacuole is a contributing factor to the immunodominance of the LLO 91-99 peptide. In Specific Aim I, studies are described that will determine if secretion of defined peptides within the phagocytic vacuole is a contributing factor to their immunodominance. For these studies, we will use L. monocytogenes mutants in which the anchor residue at position 2 within the Kd-binding nanomer peptide (normally a tyrosine) has been replaced by phenylalanine. The mutants retain the wildtype phenotype, yet following immunization with these strains, specific CTL are not stimulated to the mutant peptide sequence. We will use these mutants to deliver specific Listeria-derived peptides to the phagocytic vacuole or the cytoplasm of the L. monocytogenes infected cell in order to determine the influence of these initial locations on the subsequent CTL response. L. monocytogenes has been proposed as a vector to deliver candidate antigens and stimulate specific cell-mediated immunity. The value of vaccine vectors for delivery of candidate antigens depends in part on the ability to specifically stimulate the immune response in an environment of existing anti-vector immunity. In Specific Aim II, we will determine the utility of L. monocytogenes as a vaccine vector and assess the development of peptide specific immunity in animals that have defined levels of existing immunity to the vaccine vector. We will assess the development of peptide specific effector cell and memory cell responses. The results from these studies will guide strategies for the general development of novel vaccine carriers as well as to define the limits of their use within a population whose immunologic status to the vaccine carrier is unknown.