Shigellosis is a gastrointestinal disease of worldwide public health importance for which there is no licensed vaccine. Our group has developed a serotype-independent vaccine based on two proteins of the Shigella type three secretion system (T3SS). These proteins have important roles in pathogenesis and are conserved among virulent Shigella strains. We generated a fusion protein (DB fusion) that comprises the T3SS tip proteins IpaB and IpaD. This vaccine has been shown to be protective in the mouse pulmonary model. We propose to use the T3SS vaccine as a model to identify the host immune responses that confer protection against Shigella infection. We hypothesize that by through a thorough examination of the immune response after vaccination with the DB Fusion using parenteral and mucosal vaccination routes, two different detergents, and the lethal pulmonary and IP models in Balb/c and knockout mice, we will identify correlates of protection that can be measured in future clinical trials with this vaccine. Toward this end, the Specific Aims of this study are: 1. Determine the protective mechanism of the DB fusion vaccine in a mouse models. We will determine the type of immunity involved in protection by vaccinating animals deficient in different immune responses. B cell and several cytokine deficient animals will be vaccinated with the DB Fusion protein. Afterwards, mice will be challenged IP with S. flexneri. The immune responses identified using these experiments will be further analyzed by making transfer experiments with the appropriate cells or cytokines in order to confirm the protective mechanism;2. Determine the optimal pathway for antigen presentation. We will stimulate dendritic cells with IpaB, IpaD and the combination in the presence of dmLT and measure cytokine release and up-regulation of activation markers. The mechanism of this up-regulation will be further characterized by identifying receptor molecules in dendritic cells responsible for this response;and 3. Optimize the vaccine formulation to identify the importance of detergent in conveying protective immunogenicity. The role of detergent present in the vaccine formulation will be evaluated by analyzing the DB Fusion in presence of two different detergents. In particular we will analyze the aggregation state and general secondary structure stability of the fusion in each detergent. We will also characterize immune cells extracted from mice vaccinated with the DB Fusion with each detergent present to correlate biophysical characteristics with protective efficacy. We have assembled a team of investigators with expertise in immunology and protein biochemistry to explore the mechanism by which this novel vaccine is able to convey protection against shigellosis. In combining an identification of the correlates of protection with the use of biophysical methods for optimizing vaccine formulation, we will establish the basis for evaluating the likely efficacy of this vaccine in protecting humans against shigellosis.
Shigella flexneri is the leading bacterial cause of dysentery in the developing world and a major cause of travelers'diarrhea in the developed world. As a unique approach we have identified two proteins from the Shigella type III secretion system apparatus (T3SA) as being serotype-independent protective antigens against Shigella infections. We hypothesize that by examining of the immune response after vaccination with the DB Fusion using various vaccination routes, different formulations, and two mouse models in wild- type and knockout mice, we will identify correlates of protection that can be measured in future clinical trials with this vaccine.