Biology subjects

Gottwein, J. M.

Publications and source records attributed to Gottwein, J. M..

4 recordsLinked to original sources

bioRxiv2024

Bispecific antibodies against the hepatitis C virus E1E2 envelope glycoprotein

Hepatitis C virus (HCV) currently causes about one million infections and 240,000 deaths worldwide each year. To reach the goal set by the World Health Organization (WHO) of global HCV elimination by 2030, it is critical to develop a prophylactic vaccine. Broadly neutralizing antibodies (bNAbs) target the E1E2 envelope glycoproteins on the viral surface, can neutralize a broad range of the highly diverse circulating HCV strains and are essential tools to inform vaccine design. However, bNAbs targeting a single E1E2 epitope might be limited in neutralization breadth, which can be enhanced by using combinations of bNAbs that target different envelope epitopes. We have generated 60 IgG-like bispecific antibodies (bsAbs) that can simultaneously target two distinct epitopes on E1E2. We combine non-overlapping E1E2 specificities into three types of bsAbs, each containing a different hinge length. The bsAbs show retained or increased potency and breadth against a diverse panel of HCV pseudoparticles (HCVpp) and HCV produced in cell culture (HCVcc) compared to monospecific and cocktail controls. Additionally, we demonstrate that changes in the hinge length of bsAbs can alter the binding stoichiometry to E1E2. These results provide insights into the binding modes and the role of avidity in bivalent targeting of diverse E1E2 epitopes, and suggest structural differences between HCVpp and HCVcc. This study illustrates how potential cooperative effects of HCV bNAbs can be utilized by strategically designing bispecific constructs. These new HCV bsAbs can guide vaccine development and unlock novel therapeutic and prophylactic strategies against HCV and other (flavi)viruses.

microbiology↗

bioRxiv2022

Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro

The oral protease inhibitor nirmatrelvir is expected to play a pivotal role for prevention of severe cases of coronavirus disease 2019 (COVID-19). To facilitate monitoring of potentially emerging resistance, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir. Resistant variants selected in cell culture harbored different combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetic studies in a homologous infectious cell culture system revealed up to 80-fold resistance conferred by the combination of substitutions L50F and E166V. Resistant variants had high fitness increasing the likelihood of occurrence and spread of resistance. Molecular dynamics simulations revealed that E166V and L50F+E166V weakened nirmatrelvir-Mpro binding. The SARS-CoV-2 polymerase inhibitor remdesivir retained activity against nirmatrelvir resistant variants and combination of remdesivir and nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatment programs with high efficacy against SARS-CoV-2 and potentially emerging coronaviruses.

microbiology↗

bioRxiv2020

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

Antivirals targeting SARS-CoV-2 could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PI) against SARS-CoV-2 and their interactions with remdesivir, the only antiviral approved for treatment of COVID-19. HCV PI showed differential potency in VeroE6 cell-based antiviral assays based on detection of the SARS-CoV-2 Spike protein. Linear PI boceprevir, telaprevir and narlaprevir had 50% effective concentrations (EC50) of ~40 M. Among macrocyclic PI simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir and deldeprevir, simeprevir had the highest (EC50 15 M) and glecaprevir the lowest (EC50 >178 M) potency. Acyclic PI asunaprevir and faldaprevir had EC50 of 72 and 23 M, respectively. ACH-806, an HCV NS3 protease co-factor NS4A inhibitor, had EC50 of 46 M. For selected PI, potency was similar in human hepatoma Huh7.5 cells. Selectivity indexes, based on antiviral and cell viability assays, were highest for linear PI. In combination with remdesivir, linear PI boceprevir and narlaprevir showed antagonism, while macrocyclic PI simeprevir, paritaprevir and grazoprevir showed synergism with drug reduction indexes of up to 27 for simeprevir. Treatment of infected cultures with equipotent concentrations (1-fold EC50) of HCV PI revealed minor differences in barrier to SARS-CoV-2 escape. Complete viral suppression was achieved treating with [≥]3-fold EC50 boceprevir or combination of 1-fold EC50 simeprevir with 0.4-fold EC50 remdesivir, not leading to significant viral suppression in single treatments. Considering potency, human plasma concentrations and synergism with remdesivir, simeprevir seemed the most promising compound for optimization of future antiviral treatments of COVID-19.

microbiology↗

bioRxiv2020

Efficient culture of SARS-CoV-2 in human hepatoma cells enhances viability of the virus in human lung cancer cell lines permitting the screening of antiviral compounds

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity. ImportanceThe cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

microbiology↗