Abstracts B1 - B4

Category B.  Infectious Diseases and Vaccine Discovery


B1.  Analysis of Different Adjuvants on the Efficacy of a Subunit Vaccine in a Mouse Salmonella Model

Kelly Harrison a, Francisco J. Martinez-Becerra b, Shyamal P. Choudhari c, Olivia Arizmendi a, John D. Clements d, William D. Picking c, Wendy L. Picking c

aDepartment of Molecular Biosciences, bHiguchi Biosciences, cDepartment of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS;   dDepartment of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA.

Salmonella enterica, with over 2500 serovars, is the causative agent of nearly 1.3 billion cases of disease annually in both humans and animals. Non-typhoidal serovars such as Typhimurium cause severe gastroenteritis as well as bacteremia in children. In spite of the significant number of infections, a universal vaccine against many serovars is still absent. S. enterica possesses numerous pathogenicity islands, two of which encode type three secretion systems: SPI-1 and SPI-2.  Their vital role in pathogenicity and their highly conserved sequence among different serotypes led us to investigate the protective efficacy of the type three secretion components SipB, SipD and SseB in S. enterica infections. Mice were immunized intranasally with the SPI-1 proteins SipB and SipD, the SPI-2 protein SseB, or all three proteins combined using either monophosphoryl lipid A or double mutant heat-labile toxin (dmLT) as the adjuvant. Immunogenicity was tested through antibody titers, enumerating antibody-secreting cells and levels of cytokine secretion. Mice were then challenged with a lethal dose of S. Typhimurium administered orogastrically and up to 50% protection was observed. Our results indicate that these proteins provide protection against infection and provide information for further development of broad-range Salmonella vaccines.



B2.  Biophysical characterization and stabilization of CagL, an antigenic protein from Helicobacter pylori as a candidate subunit vaccine

Shyamal P. Choudhari1, Kirk P. Pendleton1, Joshua D. Ramsey2, Thomas G. Blanchard3, Wendy L. Picking1, and William D. Picking1

1Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS;
2Department of Chemical Engineering, Oklahoma State University, Stillwater, OK;
3Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD

A major concern associated with protein subunit vaccines is loss of activity due to physical instability that may cause aggregation of the antigen proteins. This physical instability is mainly influenced by the effect of solution conditions like pH and temperature. Excipient screening aimed at improved protein stability is an essential step to finding an ideal vaccine formulation. CagL is a type IV secretion system protein from the gram negative bacterium Helicobacter pylori. This protein is involved in attachment of the bacterium to the host cell through contact with α5β1 integrin. This role makes it a potential candidate for a subunit vaccine against H. pylori. In the present study, CagL was subjected to various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence, static light scattering and extrinsic fluorescence under different solution conditions of pH and temperature. Protein stability at each pH condition was determined in terms of transition temperature (Tm) value. The data accumulated was incorporated into a color map called empirical phase diagram (EPD) that provided an overall view of physical stability of the protein. Additionally, aggregation assays were performed to screen excipients from a library of generally regarded as safe (GRAS) compounds. Excipients that inhibited protein aggregation were chosen to confirm their enhanced stabilizing effect by the spectroscopic techniques. The increased Tm value in the presence of certain excipients was considered to be a result of improved physical stability of the protein. These data allowed us to propose the use of select compounds as potential excipients for a CagL vaccine formulation.



B3.  Characterization of immune responses during intradermal vaccination of an IpaDB fusion protein against shigellosis

Francisco J. Martinez-Becerra1, William D. Picking1 and Wendy L. Picking1‡

1Higuchi Biosciences Center, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas

Shigella spp causes around 165 million cases of bacillary dysentery every year around the world, with most of the cases occurring in children living in developing countries. There is no available vaccine for shigellosis, and the existence of more than 50 different serotypes further complicates vaccine development efforts. Our research group showed that the Type three secretion proteins IpaB and IpaD are protective antigens in a mouse model of infection. These proteins are highly conserved among all Shigella serotypes and are essential to virulence and pathogenesis. In order to optimize the vaccine formulation we generated a fusion protein that includes IpaD and IpaB in the same polypeptide chain. Furthermore we tested an intradermal delivery of this protein along the adjuvant dmLT in order to reduce the amount of protein used for immunizations. In order to better define the protective immune responses, we vaccinated mice with increasing amounts of the DB fusion with or without the adjuvant dmLT via intradermal administration. We analyzed the kinetics of antibody responses in both serum and stool samples, generation of cytokine secreting cells in spleens and activation markers in immune cells extracted from immunized animals. In addition, we challenged vaccinated animals with Shigella to evaluate the protective efficacy of our different vaccine formulations. We found that intermediate doses of the DB fusion were protective against Shigella infection. In addition, this protection was correlated with cytokine secretion profiles. These results will allow us to further optimize our vaccine candidate for intradermal delivery aiming to start clinical trials.



B4.  The Shigella flexneri T3SS tip protein IpaD elicits caspase-dependent cell death in macrophages

Olivia Arizmendi1, Francisco J. Martinez Becerra1, William D. Picking1 and Wendy L. Picking1‡

1Higuchi Biosciences Center, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047

Shigellosis, a type of bacillary dysentery, is a gastrointestinal infectious disease caused by Shigella spp. Approximately 165 million cases of shigellosis are reported every year around the world, most of them in developing countries. Shigella is able to colonize the human colonic epithelium and promote an inflammatory response only after inducing cell death of residing macrophages. All of these virulent actions are intimately tied to the Type III Secretion System (T3SS) of this organism. Invasion plasmid antigen D (IpaD) is a structural element at the tip of the needle that controls secretion of effectors to alter host cell functions. IpaD has also been shown to induce cell death via apoptosis in B lymphocytes. We performed cytotoxicity, caspase activation and inhibition assays as well as a multiplex approach to study the cytokine response to IpaD. Furthermore, a possible binding partner was found. Together, this data allows us to conclude that IpaD is also able to elicit apoptosis in macrophages and more studies are needed to show the pathways that could enable IpaD to trigger these pro-apoptotic signals in macrophages.


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