Category B. Infectious Diseases and Vaccine Discovery
B1. Assessment of Serotype-independent Immunity Elicited by Shigella T3SS Proteins
Francisco J. Martinez-Becerra, Melissa Pressnall, Olivia Arizmendi Perez, William D. Picking, Wendy L. Picking
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA
Shigellosis is a gastrointestinal disease of worldwide public health importance for which there is no licensed vaccine. It is also classified as a category B bioterrorism agent. 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 using vaccine/infection models we will identify correlates of protection that can be measured in future clinical trials with this vaccine. We determined the type of immunity involved in protection by vaccinating animals using different administration routes. Afterwards, mice are challenged with S. flexneri. We also stimulated dendritic cells with IpaB, IpaD and the combination in the presence of dmLT and measure cytokine release and up-regulation of activation markers. In combining an identification of the correlates of protection with the use of biophysical methods for optimizing vaccine formulation, we hope to establish the basis for evaluating the likely efficacy of this vaccine in protecting humans against shigellosis.
B2. Activation of Dendritic Cells by the subunit vaccine for Shigella
Melissa Pressnall1, Francisco Martinez-Becerra1, Olivia Arizmendi-Perez2, Wendy Picking1, William Picking1
1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA; 2Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Shigellosis is a disease with severe global impact yet it has no approved vaccine. This gastrointestinal disease effects ~90 million people per year with 100,000 deaths primarily in young children in developing countries. Shigella infects humans by utilizing a type three secretion system (T3SS) which is highly conserved over the ~50 Shigella serotypes. The T3SS works like a needle and syringe that injects bacterial protein effectors into the host epithelial cells in the colon to promote bacterial entry. The internalized bacteria then lyse the resulting phagosome which allows bacterial replication in the host cell cytoplasm. Two proteins, IpaB and IpaD, control the activity of the T3SS from the tip of the needle. Using a lethal pulmonary mouse challenge model, we have shown that vaccination with IpaB + IpaD or a genetic fusion of these proteins (the DB Fusion) along with the adjuvant dmLT is protective against a Shigella flexneri challenge as well as a heterologous challenge by Shigella sonnei. In addition, we found that using both antigens (either combined or in the DB Fusion) gives rise to higher cytokine activation and protection profiles. We are working to determine the protective mechanism of the DB Fusion vaccine and to identify the pathway for antigen presentation. The first step in this effort is to analyze the effect of these proteins in antigen presenting cells such as dendritic cells. In this study, we are stimulating dendritic cells with IpaB, IpaD, IpaB+IpaD, or the DB Fusion, with or without dmLT to measure subsequent cytokine release as well as the up-regulation of activation markers. Identification of the activation markers and cytokines involved will help to describe a plausible mechanism for the response of dendritic cells incubated with our candidate vaccine proteins.
B3. Development of β–Barrel Ligands for Antibiotic Potentiation
Pinakin Sukthankar1, Cyril Cook1, Amritangshu Chakravarty1, and Joanna Slusky1,2
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA; 2Center for Computational Biology, University of Kansas, Lawrence, KS, USA
Antibiotic resistance portends an increasingly critical global health challenge. The fatalities caused by antibiotic resistant bacterial infections are estimated to surpass those of cancer by 2050. Antibiotic resistance is correlated with the overexpression of the acridine efflux pump. This pump is the preeminent efflux pump in gram-negative bacteria and is responsible for shuttling out most classes of antibiotics. The outer membrane component of the acridine Efflux pump is a trimeric beta barrel called TolC. Our central hypothesis is that TolC oligomerization can be disrupted through a dominant-negative, fragment-based inhibitor. Or goal is to disrupt TolC oligomerization, stop bacterial efflux, and potentiate antibiotics.
B4. Assessment of Protective Efficacy of a Broadly Protective Subunit Vaccine to Prevent Infection by Salmonella enterica
Vikalp Vishwakarma1, Francisco J. Martinez-Becerra1, Prashant Kumar1, Olivia Arizmendi2, Melissa M. Pressnall1, William D. Picking1,3, Wendy L. Picking1
Departments of Pharmaceutical Chemistry1 and Molecular Biosciences2, Higuchi Bioscience Center3, University of Kansas, Lawrence, KS, USA
Infections from non-typhoidal Salmonella enterica (NTS) is the leading cause of sickness and death in US. It infect livestock and thus a serious threat to the food animal industry and human health. Development of broadly protective vaccines against S. enterica serovars is an important need and a field of ongoing research. Here we developed a potential vaccine in which SPI-1 and SPI-2 tip and first translocator proteins, SipB/SipC and, SseB/SseC, respectively, were genetically fused to produce recombinant fusion proteins S1 and S2, respectively, for immunization of mice. S1 and S2, alone or together, were administered intramuscularly with monophosphoryl lipid-A (MPL) and Alhydrogel as adjuvant system. Immunization elicited a high serum IgG response against both S1 and S2 protein antigens. Highest frequencies of IgG antibody secreting cells (ASCs) were measured from the bone marrow of mice immunized with S1S2 combination; however, immunization with S1 and S2 alone generated moderate to high frequencies of ASCs, respectively. Similarly, unique cytokine secretion patterns were detected in S1S2 vaccinated mice. Upon lethal challenge with S. Typhimurium or S. Enteritidis, mice group vaccinated with S1S2 formulation elicited the highest protection against death. S1S2 immunization prevented mice cecal inflammation as compared to immunization with S1 and S2 alone. These results demonstrate the proof of concept in a small animal model that S1S2 subunit vaccine can provide broad coverage to protect against all S. enterica serovars which may be transformative to the livestock industry and improve human health.