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Browsing by Author "Glass, Pamela J."

Now showing 1 - 3 of 3
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    Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis
    Liu, Jinny L.; Zabetakis, Dan; Gardner, Christina L.; Burke, Crystal W.; Glass, Pamela J.; Webb, Emily M.; Shriver-Lake, Lisa C.; Anderson, George P.; Weger-Lucarelli, James; Goldman, Ellen R. (Nature Portfolio, 2022-01-13)
    Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.
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    In Silico Screening of Inhibitors of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease
    Hu, Xin; Morazzani, Elaine; Compton, Jaimee R.; Harmon, Moeshia; Soloveva, Veronica; Glass, Pamela J.; Garcia, Andres Dulcey; Marugan, Juan J.; Legler, Patricia M. (MDPI, 2023-07-04)
    The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester (CA074me) and a reversible oxindole inhibitor. Here, we determined the X-ray crystal structure of the CA074-inhibited nsP2 protease and compared it with our E64d-inhibited structure. We found that the two inhibitors occupy different locations in the protease. We designed hybrid inhibitors with improved potency. Virus yield reduction assays confirmed that the viral titer was reduced by >5 logs with CA074me. Cell-based assays showed reductions in viral replication for CHIKV, VEEV, and WEEV, and weaker inhibition of EEEV by the hybrid inhibitors. The most potent was NCGC00488909-01 which had an EC50 of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC50 = 7.90 µM. Other compounds from the NCATS libraries such as the H1 antihistamine oxatomide (>5-log reduction), emetine, amsacrine an intercalator (NCGC0015113), MLS003116111-01, NCGC00247785-13, and MLS00699295-01 were found to effectively reduce VEEV viral replication in plaque assays. Kinetic methods demonstrated time-dependent inhibition by the hybrid inhibitors of the protease with NCGC00488909-01 (Ki = 3 µM) and NCGC00484087 (Ki = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl2, MnCl2, or MgCl2 were measured with varying concentrations of inhibitor, Mg2+ and Mn2+ slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV.
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    Stabilization of a Broadly Neutralizing Anti-Chikungunya Virus Single Domain Antibody
    Liu, Jinny L.; Webb, Emily M.; Zabetakis, Dan; Burke, Crystal W.; Gardner, Christina L.; Glass, Pamela J.; Legler, Patricia M.; Weger-Lucarelli, James; Anderson, George P.; Goldman, Ellen R. (2021-01-28)
    A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values similar to 1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10 degrees C over the original CC3 (T-m = 62 degrees C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.