Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.

Reduced Neutralization of SARS-CoV-2 Omicron Variant by Vaccine Sera and monoclonal antibodies

Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.

Characterization of the humoral immune response to BNT162b2 in elderly residents of long-term care facilities five to seven months after vaccination

The elderly residing in long-term care facilities (LTCFs) are a group at high risk for COVID-19. Hence, monitoring of the vaccine-based immunity has a pivotal role in identifying strategies to provide optimal protection in this population. We examined the immune response to the mRNA vaccine BNT162b2 against COVID-19 five to seven months after completing a two-dose regimen. We determined significantly lower anti-SARS-CoV-2 antibody titers in 298 SARS-CoV-2 naive residents who were at least 75 years of age (mean 51.60 BAU/ml) (median age 87 years, range 75 to 101 years) when compared to health care workers (HCWs) aged 18 to 70 years (mean 156.99 BAU/ml, p < 0.001). Of the SARS-CoV-2 naive residents, 29 had detectable neutralizing antibodies against the Delta variant (9.5%), and 14 of those (48.3%) only had a borderline titer of 1:10. Of 114 HCWs, 36 (31.6%) had detectable neutralizing antibodies. In a group of 14 elderly residents who had had a PCR-confirmed breakthrough infection, the mean antibody titer was significantly higher than in the other two groups (3199.65 BAU/mL) (p < 0.001), and 12 (85.7%) had detectable neutralizing antibodies against the Delta variant. Our data demonstrate that 90.5% of elderly residents of LTCFs had no detectable neutralization-competent antibodies against the dominant Delta variant five to seven months after vaccination, and that neutralizing antibody titers were restored following a break-through infection. Our results suggest that both residents and health care workers in LTCFs would benefit from a booster vaccine six months after completing the two-dose schedule or earlier.

Antibody mediated neutralization of authentic SARS-CoV-2 B.1.617 variants harboring L452R and T478K/E484Q

The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.5-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which however, might be circumvented by a combination therapy with casirivimab together.

Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

At-home self-testing of teachers with a SARS-CoV-2 rapid antigen test to reduce potential transmissions in schools

BackgroundRapid antigen tests for SARS-CoV-2 became available recently, offering an opportunity to vastly increase testing capacities. Antigen tests offer lower sensitivity than the gold standard, RT-PCR, but rapid sample-to-answer time. High-frequency testing with an antigen test may offset the lower sensitivity, and testing can be done with at-home collection of samples, offering potential benefit in screening efforts. In this study, we set out to evaluate the practical application of self-performed high-frequency antigen test in a school setting. MethodA total of 711 teachers from 86 schools were enrolled in a seven-week study. After instruction, participants tested themselves every 48 hours at home with a rapid antigen test for SARS-CoV-2 (target: nucleocapsid protein) in a self-collected anterior nasal swab. Positive results in the antigen test were confirmed via RT-PCR from the same sample that had been determined to be positive by the study participant. A questionnaire was given to all participants to evaluate whether the test failed to detect infection. Findings10 836 tests from 602 teachers were recorded and analyzed. A total of five confirmed cases of viral shedding of SARS-CoV-2 was detected by use of the antigen test. One study participant with a SARS-CoV-2 infection was presymptomatic and four were mildly symptomatic at the time of the antigen test. Sixteen false positive antigen tests (0.15% of all tests) were reported, predominantly when the local incidence in the general population was low. In four cases, the study participant reported that a PCR had detected a SARS-CoV-2 infection, but the antigen test was negative, indicating a false negative result. InterpretationHigh-frequency, self-performed rapid antigen tests can detect individuals with a SARS-CoV-2 infection, and therefore potentially reduce transmissions. Testing may be most beneficial when applied during high local incidence of SARS-CoV-2 infections and when mild or atypical symptoms are present. To avoid a high rate of false positive results, a test with optimized specificity should be used. FundingThe study was commissioned and funded by the Hessian Ministry of Education and the Hessian Ministry of Integration and Social Affairs.

Evaluation of a SARS-CoV-2 rapid antigen test: potential to help reduce community spread?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from symptomatic patients with COVID-19, but also from asymptomatic individuals. Therefore, robust surveillance and timely interventions are essential for the control of virus spread within the community. In this regard the frequency of testing and speed of reporting, but not the test sensitivity alone, play a crucial role. In order to reduce the costs and meet the expanding demands in real-time RT-PCR (rRT-PCR) testing for SARS-CoV-2, complementary assays, such as rapid antigen tests, have been developed. Rigorous analysis under varying conditions is required to assess the clinical performance of these tests and to ensure reproducible results. We evaluated the sensitivity and specificity of a recently licensed rapid antigen test using 137 clinical samples in two institutions. Test sensitivity was between 88.2-89.6% when applied to samples with viral loads typically seen in infectious patients. Of 32 rRT-PCR positive samples, 19 demonstrated infectivity in cell culture, and 84% of these samples were reactive with the antigen test. Seven full-genome sequenced SARS-CoV-2 isolates and SARS-CoV-1 were detected with this antigen test, with no cross-reactivity against other common respiratory viruses. Numerous antigen tests are available for SARS-CoV-2 testing and their performance to detect infectious individuals may vary. Head-to-head comparison along with cell culture testing for infectivity may prove useful to identify better performing antigen tests. The antigen test analyzed in this study is easy-to-use, inexpensive, and scalable. It can be helpful in monitoring infection trends and thus has potential to reduce transmission.

Optimized qRT-PCR approach for the detection of intra- and extracellular SARS-CoV-2 RNAs

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.