Molecular Microbiology and Immunology presentations (MU)
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Items in this collection are public presentations made by Department of Molecular Microbiology and Immunology faculty, staff, and students, either alone or as co-authors, and which may or may not have been published in an alternate format. Items may contain more than one file type.
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Item The Antigen Display on Bacillus Endospore (ADOBE) System a Noninvasive Biodegradable Microparticle Display System(2010) Pritzl, C. J.; Hassett, Daniel E.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)The development of safe and effective vaccines and adjuvants remains an important global public health goal. The Antigen Display on Bacillus Endospore (ADOBE) system, developed at the University of Missouri's College of Veterinary Medicine, is a unique, non-replicating, microparticle-based antigen delivery platform with inherent adjuvant properties. Killed spores can be readily engineered to present single or multiple antigens to the immune system. Bioactive targeting molecules and molecular adjuvants can also be co-displayed with the immunogen on the spore surface to enhance specific innate or acquired immune responses. The combination of a strong natural adjuvant and an easily produced microparticle delivery vehicle makes ADOBE-based vaccines excellent candidates for preclinical development against a large number of human and veterinary diseases. Because virtually any molecule of interest can be covalently attached to the outer spore surface, the ADOBE method also allows for the use of spores as biodegradable solid-phase platforms for use in diagnostic tests, molecular imaging, biocatalytic reactions, and the identification, quantification, and or purification of specific compounds from a complex mixture of compounds. We are currently looking for corporate as well as academic collaborators that are interested in capitalizing on the ADOBE methodology for the development of novel biopharmaceuticals to diagnose, treat and prevent infectious and metastatic diseases.Item Novel inhibitors of Foot and Mouth Disease Virus (FMDV) Targeting the RNA-Dependent RNA Polymerase activity of 3Dpol [abstract](2010-03) Durk, Ryan C.; Schafer, Elizabeth; Moran, Jennifer L.; Singh, Kamlendra; Marchand, Bruno; Michailidis, Eleftherios; Adedeji, Adeyemi; Pautler, Christine M.; Rodriguez, Luis; McIntosh, Mark A.; Rieder, Elizabeth; Sarafianos, Stefan G.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Foot-and-Mouth Disease Virus (FMDV) is a positive stranded picornavirus which infects cloven-hoofed animals, such as cattle, pigs and sheep, and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination could be used but it would require at least 7 days to trigger an effective immune response. On the contrary, the use of antiviral drugs is expected to have prophylactic and/or therapeutic effects almost immediately. However, there are currently no approved FMDV inhibitors. Here we have applied a combination of screening, biochemical, virological, and molecular modeling tools to discover, validate, and characterize novel inhibitors of FMDV replication. Using a luciferase-based assay we have screened a chemical library of compounds and have identified two compounds, 5-chloro-3-(thiophen-2-yl-sulfanylmethyl)-1-benzothiophene 1,1-dioxide (or C7F8) and N'1-thieno[2,3-d]pyrimidin-4-yl-4-chloro-1-benzenesulfonohydrazide (or C5D9) that inhibited the RNA-dependent RNA polymerase activity of FMDV replicase (3Dpol) with IC50 values of 2.5 μM and 15 μM respectively. These compounds were shown to be specific inhibitors of FMDV 3Dpol and not nucleic acid chelators, as they did not affect activity of other viral polymerases using the same nucleic acid substrate. Molecular modeling docking experiments suggest that both inhibitors bind at a pocket proximal to, but distinct from, the NTP binding site of 3Dpol, thereby affecting indirectly RNA synthesis. C7F8 and C5d9 were not cytotoxic at concentrations up to at least 100 ||M. Importantly, C5D9 exhibited antiviral activity and suppressed virus production in FMDV-infected cells with 50% and 90% effective concentrations (EC50 and EC90) of 10 ||M and 20 ||M, respectively. The results indicate that 3Dpol inhibitors can be promising anti-FMDV agents for use as alternative or supplementary options to contain future outbreaks of FMD.Item Discovery and Mechanistic Characterization of Novel SARS Coronavirus Inhibitors that Block Viral Entry [abstract](2010-03) Adedeji, Adeyemi; Severson, William; Jonsson, Colleen; Michailidis, Eleftherios; Marchand, Bruno; Singh, Kamlendra; Weiss, Susan R.; Sarafianos, Stefan G.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS-associated coronavirus (SARS-CoV). Viral entry is a key target step for therapies because it can prevent the propagation of virus at early stages of the disease. We used a cell-based assay to identify inhibitors of SARS-CoV entry. We prepared a pseudotyped virus in which the core is from HIV and envelop is from the SARS-CoV (HIV-luc/SARS env). This pseudotyped virus was used to infect, 293T cells expressing the receptor for SARS-CoV, Aangiotensin-converting enzyme-2 (ACE2). Using this assay we screened a chemical library of more than 2000 compounds and identified three compounds that specifically inhibit entry of the HIV-luc/SARS env. These compounds did not inhibit another pseudotyped virus which had same core from HIV but envelop was from Vesicular Stomatitis Virus. The compounds had strong potencies (EC50s were 2.9, 4.8 and 5.8 µM) and low cytotoxicities (high CC50s) resulting in promising Selectivity Indices (CC50/EC50 were >175, >65, and >86, respectively). Importantly, the compounds were found to have excellent antiviral activities, blocking SARS-CoV replication at low nM concentrations. Only one of the compounds was a moderate inhibitor of cathepsin L, a cellular protease whose activity is required to process the SARS-CoV env glycoprotein (Spike) and allow viral entry. Moreover, none of the compounds affects the cleavage activity of furin, another host protease, which may also be involved in SARS-CoV entry. Using a flow cytometry binding assay, we found that all three compounds decrease binding of the SARS-CoV Spike receptor binding domain to ACE2 receptor expressed on the surface of 293Tcells. Hence, we have discovered three promising compounds as the first small molecule inhibitors that can block receptor-dependent entry of SARS-CoV.Item Inhibition Mechanism of EFdA, a Highly Potent Inhibitor of HIV Reverse Transcriptase [abstract](2010-02) Michailidis, Eleftherios; Marchand, Bruno; Kodama, Eiichi N.; Singh, Kamlendra; Kirby, Karen, 1979-; Ong, Yee T.; Ryan, Emily M.; Nagy, Eva; Ashida, Noriyuki; Murphey-Corb, Mickey; Mitsuya, Hiroaki; Parniak, Michael A.; Sarafianos, Stefan G.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)The nucleoside 4'-ethynyl-2-fluoro-deoxyadenosine (EFdA) is one of the most potent antiretroviral nucleosides yet described, inhibiting replication of wild-type and multidrug-resistant HIV-1 strains in vitro (PBMC cells) with an EC50 as low as 50 pM. Our laboratory works in collaboration with academic, government and pharmaceutical industry laboratories, to characterize the mechanism of action of EFdA, and help develop it as a therapeutic for the treatment of HIV-infected patients, and as a topical microbicide to minimize sexual transmission of HIV. We have recently shown that the potency of antiviral activity stems in part from a mechanism of action not shown by any of the clinically used nucleoside antiretrovirals. Unlike other Reverse Transcriptase (RT) inhibitors, EFdA has a 3'-OH group which is necessary for nucleotide incorporation, yet it acts as a chain-terminator of retroviral DNA synthesis. Using biochemical techniques, we have determined that EFdA is incorporated very efficiently into the nascant viral DNA chain and blocks the incorporation of incoming nucleotides by stopping the translocation/movement of RT. Therefore, we have dubbed EFdA as a Translocation-Defective RT Inhibitor (TDRTI). A pilot collaborative study spearheaded by collaborators Parniak and Corb demonstrated that EFdA treatment of Rhesus Macaques resulted in a 2-3 log decrease in simian immunodeficiency virus (SIV) within seven days; these levels declined to undetectable levels (5-log reduction) within 2 months and essentially remained so for the duration of therapy. Hence, EFdA is a highly potent HIV RT inhibitor with in vitro and in vivo antiviral activities that warrant further development of the compound as a potential therapeutic for individuals harboring wild-type and/or multi-drug resistant HIV-1.Item High Resolution Crystal Structure of KD-247, a Humanized Antibody that Inhibits HIV Entry [abstract](2010-02) Kirby, Karen, 1979-; Moran, Jennifer L.; Marchand, Bruno; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Highly active antiretroviral therapy (HAART) has been very efficient in reducing the rate of mortality of human immunodeficiency virus type 1 (HIV-1) infected patients. However, resistance to clinically used drugs inevitably develops and impairs the potency of these drugs. There is also no vaccine available to prevent the spread of the virus. Our collaborators, Dr. Shuzo Matsushita and his colleagues have developed a monoclonal antibody, KD-247, that is currently in Phase Ib clinical trials for the treatment of HIV-1 infections. KD-247 blocks virus entry into host cells by binding to the V3 loop of the surface glycoprotein of HIV. It is the first humanized antibody shown to neutralize a wide range of subtype B HIV viruses (Matsushita et al. Hum. Antibodies, 14, 81) and to prevent HIV infection in cell culture and in a chimpanzee model (Eda et al. J. Virol., 80, 5563). KD-247 reacts exclusively with subtype B viruses (Eda et al. J. Virol., 80, 5552). In order to understand the molecular basis of this specificity we have solved the crystal structure of KD-247 at 1.5 Å resolution, the highest resolution structure for any humanized antibody reported to date. The present structure reveals in atomic detail the molecular boundaries of a pocket formed by the antigen-binding region of the antibody. Molecular docking experiments of a pre-existing structure of the V3-loop target at the presumed binding pocket on KD-247 suggest possible molecular interactions involved in HIV resistance to KD-247 and clade B specificity. A G314E V3 loop mutation that has been reported to confer resistance to KD-247 (Yoshimura et al., AIDS 20, 2065) appears to result in steric interactions between the tip of the V3 loop and residues of the heavy chain of KD-247. Further, Arg315, a residue critical for clade B specificity, appears to form extensive interactions with multiple residues of KD-247. Analysis of these interactions has provided insights into the design of second-generation antibodies with broader subtype specificity and improved ability to evade resistance mutations. This work is a product of collaborations between the University of Missouri and researchers at Kumamoto University, an academic institution in Japan, and the Chemo-Sero-Therapeutic Research Institute, an industrial partner in Japan, working to commercialize the antibody and further its progress in clinical trials.