Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology (preprint)

The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology and titers were comparable between rescued molecular clones and a clinical isolate (VIDO strain), thus providing confidence that the rescued viruses can be used as effective replication tools. Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells, were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death and a novel lung cell line NCI-H23ACE2 A3 showed about ~99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection and are currently being used in CRISPR knockout and activation screens in our lab.

Cancer Therapy and Immunogenicity of COVID Vaccine – CANINE Study (preprint)

Background: Information regarding antibody response to COVID-19 vaccines in cancer patients undergoing therapy is needed for vaccine recommendations and timing of additional doses. This longitudinal study evaluates anti-RBD and neutralizing antibody (NAb) response to COVID-19 vaccines in patients on treatment and post stem cell transplant (SCT).Methods: For mRNA vaccines, anti-RBD and NAb were assessed before vaccination (T0), prior to 2nd dose (T1), 1 month after the 2nd dose (T2), and 3 months after the 2nd dose (T3). For J&J, T1 was 1 month, T2, 2 months and T3, 4 months after vaccine. Primary objective was GMTs of anti-RDB and NAb (%) at above timepoints. Other objectives were a) proportion of patients with anti-RBD ≥100 U/mL, b) correlation between anti-RBD and NAb, c) antibody responses of the 2 mRNA vaccines, and d) anti-RBD in breakthrough COVID-19 cases.Findings: Between 3/2/2021 and 7/30/2021, 438 cancer patients were enrolled. 108 (25%) were post-SCT and 330 (75%) on treatment: 176 (40%) on chemotherapy (C), 21 (5%) on chemoimmunotherapy (C+I), 72 (19%) on immunotherapy (I), and 58 (13%) on targeted oral agents (TOA). 60 % received Pfizer, 36 % Moderna, and 4% J&J. 11·82% of patients had anti-RBD ≥100 U/mL at T0, 25·15% at T1, 75·44 % at T2, and 81·38% at T3. At T3, 84·91% of patients on C, 81·89% on C+I, 86·67% on I, 78·18% post-SCT, and 77·50% on TOA had anti-RBD ≥ 100 U/mL. GMTs were 1·59 at T0, 12·91 at T1, 480·8 at T2 and 439·1 at T3. Neutralization (≥30%) was observed in 14·71%, 38·89%, 80·56% and 81·33% of patients at T0, T1, T2, and T3. There was no difference between the mRNA vaccines. Five patients had breakthrough infection. Four with anti-RBD available had pre-infection anti-RBD <100 U/mL. Interpretation: Four months after SARS-CoV-2 vaccination, ~ 80% of patients on cancer therapy or post-SCT have anti-RBD ≥100 U/mL and ≥30% NAb. Anti-RBD ≥100 U/mL predicts virus neutralization with accuracy. In regions with limited vaccine availability/hesitancy, antibody testing can identify 20% of the patients with relatively low titres for booster prioritization..Funding Information: This work was supported in part by the University of Kansas Cancer Centre and the Investigator Initiated Steering Committee, a grant from the NIGMS (P20 GM130423), and The University of Kansas Cancer Centre Support Grant from the NCI (P30 CA168524). A.K.G. is the Chancellors Distinguished Chair in Biomedical Sciences Endowed. Declaration of Interests: AKG and ZYP are co-founders of Sinochips Diagnostics; PS serves as advisory board member and consultant to Merck, Novartis, Exact Sciences, Seattle Genetics, Immunomedics, Myriad Genetics, AstraZeneca, Puma Biotechnology; JZ served as a scientific advisor/consultant for AstraZeneca, Biodesix, Novocure, Bayer, Daiichi Sankyo, Mirati, Novartis, Cardinal Health, Bristol Myers Squibb, Nexus Health and Sanofi and is on the speakers’ bureau for AstraZeneca and MJH Life Sciences and has received research funding from AstraZeneca, Biodesix, Novartis, Genentech/Roche, Mirati, AbbVie and Hengrui Therapeutics; GCD serves on an advisory board for Novartis; JPM serves as advisory board member and consultant to Novartis, Kite Pharmaceuticals, BMS and Allovir; RAR received institutional support from Bayer, NuCana, Incyte, AstraZeneca, Eureka therapeutics, Merck, Pfizer, and owns stock in Seattle Genetics, Actinium Pharmaceuticals Inc.; MH received consulting fees from Janssen, Pharmacyclics, Novartis Inc., Kite Pharmaceuticals and TG therapeutics. The remaining authors declare no competing interests.Ethics Approval Statement: This study was approved by the Institutional Review Board of the University of Kansas Medical Centre.