This application will bring together the expertise of different investigators and incooperate state of the art platform technologies with the goal to optimize monoclonal antibodies (mAb) to Staphylococcal enterotoxin B (SEE) for human use. Beyond the immediate benefit of yielding a novel therapeutic reagent, this work will investigate several hypotheses that pertain to toxin-neutralization and clearance. The underlying working hypothesis of our proposal is that high affinity anti-SEB mAbs are best suited for therapeutic use because low amounts of mAb will be sufficient to successfully compete with already bound SEB. We will investigate if human mAbs differ and are superior in their ability to neutralize toxin, In addition we propose that targeted engineering of the C-terminal with effects on FcR binding will change toxin clearance in vivo. In the past funding period 11 mAbs to SEB have been subcloned, characterized and tested in murine animal model for SEB induced shock. A highly sensitive capture ELISA was developed that will allow us in proposed experiments to measure SEB toxin in serum of intoxicated mice. In addition the prevalance of SEB producing S. aureus strains has been established and identified important sequence variabilities in different S. aureus strains which will be taken into consideration. Three lgG1 candidate mAbs have been selected for targeted Ab engineering. They recognize epitopes in the C-terminal part of the protein, which is also the predominant epitope recognized by human B-cells. Protection was documented in animal models and human T-cell stimulation assays. In addition heavy and light chain variable regions of the mAbs were cloned.
In Aim one we propose to optimize these mAbs by affinity maturation in collaboration with Dr. Scharff.
In Aim 2 we propose to generate new human SEB apecific mAbs one involving a genetically modified mouse that expresses the human variable region and the other one we will use Drs. Ahmed/Wilson's technique to directly clone the variable region from patients with primary S. aureus infection. Finally in aim 3 we will engineer the FC binding portion of the mAbs in collaboration with Dr. Ravetch and test the intriguing hypothesis that combination of mAbs enhances neutralization and clearance.
This proposal fulfills a major goal of this RFA because it will yield optimized SEB-specific mAbs that can be used to treat exposed humans. Optimization is expected to improve Ab efficacy, allow treatment with a lower dose and therefore lower cost. But beyond the immediate benefit of a novel therapeutic reagent this work will investigate hypothesis that pertain to toxin-mAb binding and toxin-mAb clearance. Thus knowledge gained from the proposed experiments will be valuable for the design of other mAb based treatment regimens especially those that lack good animal models.
|Johnson, Joshua C; Martinez, Osvaldo; Honko, Anna N et al. (2014) Pyridinyl imidazole inhibitors of p38 MAP kinase impair viral entry and reduce cytokine induction by Zaire ebolavirus in human dendritic cells. Antiviral Res 107:102-9|
|Gough, Daniel J; Marié, Isabelle J; Lobry, Camille et al. (2014) STAT3 supports experimental K-RasG12D-induced murine myeloproliferative neoplasms dependent on serine phosphorylation. Blood 124:2252-61|
|Kazakov, Teymur; Kuznedelov, Konstantin; Semenova, Ekaterina et al. (2014) The RimL transacetylase provides resistance to translation inhibitor microcin C. J Bacteriol 196:3377-85|
|Rajsbaum, Ricardo; Garcia-Sastre, Adolfo; Versteeg, Gijs A (2014) TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity. J Mol Biol 426:1265-84|
|Lo, Michael K; Bird, Brian H; Chattopadhyay, Anasuya et al. (2014) Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters. Antiviral Res 101:26-9|
|Mackow, Erich R; Dalrymple, Nadine A; Cimica, Velasco et al. (2014) Hantavirus interferon regulation and virulence determinants. Virus Res 187:65-71|
|Varshney, Avanish K; Wang, Xiaobo; Aguilar, Jorge L et al. (2014) Isotype switching increases efficacy of antibody protection against staphylococcal enterotoxin B-induced lethal shock and Staphylococcus aureus sepsis in mice. MBio 5:e01007-14|
|Chung, Lawton K; Philip, Naomi H; Schmidt, Valentina A et al. (2014) IQGAP1 is important for activation of caspase-1 in macrophages and is targeted by Yersinia pestis type III effector YopM. MBio 5:e01402-14|
|DiLillo, David J; Tan, Gene S; Palese, Peter et al. (2014) Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc?R interactions for protection against influenza virus in vivo. Nat Med 20:143-51|
|Szaba, Frank M; Kummer, Lawrence W; Duso, Debra K et al. (2014) TNF? and IFN? but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection. PLoS Pathog 10:e1004142|
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