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

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.

Characterization of antibody and T-cell response after second booster vaccination (preprint)

The recent surge of infections with SARS-CoV-2 Omicron subvariants of prompted countries, such as Israel and Germany, to call for an accelerated booster vaccination program for health care workers and vulnerable groups in order to limit disease and transmission. However, detailed studies analyzing the correlates of protection over time after second booster vaccination are still lacking. Here, we examined the production of Spike receptor binding domain (RBD) -specific antibodies as well as neutralizing antibodies from subjects before, two, and seven weeks after the second booster vaccination against the D614G harboring B.1 variant as well as the variants of concern (VOC) Alpha, Beta, Delta in addition to Omicron BA.1 and BA.2. The second booster vaccination resulted in an increase in anti-RBD IgG antibodies and neutralizing antibodies against B.1 in all individuals tested, then remained nearly constant over the observed period. In addition, a 2nd booster resulted in an increase in neutralizing antibodies against VOCs Alpha, Beta, Delta, and Omicron subvariants BA.1 and BA.2. However, compared to B.1 the neutralizing capacity of both Omicron subvariants remained low. Neutralization of Omicron BA.1 and BA.2 was limited even after the 2nd booster vaccination indicating that an antibody-mediated protection against infection with this VOC is unlikely, as evidenced by the fact that three of the quadruple vaccinated individuals became infected with BA.1 during the course of the study. Moreover, T cell activation measured by interferon gamma release was detected in all subjects after the 2nd booster vaccination. This may offer protection suggesting protection against severe disease. T-cell activation was independent of the age of the subjects, but correlated with the amount of Spike-specific antibodies. Interestingly, in subjects with Omicron BA.1 breakthrough infection, a significant increase in neutralizing antibodies to all tested VOCs studied was observed after the 2nd booster vaccination. Taken together, our data suggest inferior protection from breakthrough infection with the Omicron subvariant BA.1 when compared to other VOCs after four vaccine doses.

COVID-19 in a Multiple Myeloma Patient: Cellular and Humoral Immunity Against SARS-CoV-2 in a Short- and Long-term View (preprint)

Multiple myeloma patients are often treated with immunomodulatory drugs, proteasome inhibitors or monoclonal antibodies until disease progression. Chronic therapy in combination with the underlying disease frequently results in severe humoral and cellular immunodeficiency, which often manifests in recurrent infections. Here we report on the clinical management and immunological data of one multiple myeloma patient diagnosed with COVID-19. Despite severe hypogammaglobulinemia, deteriorated T cell counts and neutropenia, the patient unexpectedly combated COVID-19 by balanced response of innate immunity, strong CD8+ and CD4+ T cell activation and differentiation, development of specific T-cell memory subsets, as well as development of anti-SARS-CoV-2 type IgA and IgG antibodies. Even 6 months after re-introduction of lenalidomide maintenance therapy, specific T cell response and antibody levels remained detectable, indicating persisting immunity against SARS-CoV-2. We conclude that in MM patients who tested positive for SARS-CoV-2 and were receiving active MM treatment, immune response assessment could be a useful tool to help guide decision-making regarding the continuation of anti-tumor therapy and supportive therapy.

Longitudinal Testing of Respiratory and Gastrointestinal Shedding of SARS-CoV-2 in Day Care Centres in Hesse, Germany: Results of the SAFE KiDS-Study (preprint)

Background: With the pandemic of SARS-CoV-2 ongoing in Europe in July of 2020, day care centres were reopened in the state of Hesse, Germany, after the lockdown. The role young children play in the dynamics of the transmission was unknown.Methods: We conducted a longitudinal study over a period of 12 weeks (18th of June 2020 to 10th of September, 2020) to screen attendees and staff from day care centres in the state of Hesse, Germany, for both respiratory and gastrointestinal shedding of SARS-CoV-2. 825 children (age range 3 months to 8 years) and 372 staff members from 50 day-care centres, which were chosen representatively from throughout the state, participated in the study. Parents were asked to perform both a buccal mucosa and an anal swab on their children once a week. Staff were asked to self-administer the swabs. RT-PCRs for SARS-CoV-2 were performed in a multiple-swab pooling protocol.Findings: 7,366 buccal mucosa swabs and 5,907 anal swabs were analysed. No respiratory or gastrointestinal shedding of SARS-CoV-2 was detected in any of the children. Shedding of SARS-CoV-2 could be detected in two staff members from distinct day care centres. Case 1 was a 37 year old female who was asymptomatic at the time of testing. The second case was a 63 year old female who was symptomatic.Interpretation: Respiratory or gastrointestinal shedding of SARS-CoV-2 in children or staff members in day-care centres was very rare in the context of low community activity. The data indicate day care centres do not pose a reservoir for SARS-CoV-2 in a low prevalence setting, no inapparent transmissions were observed.Funding Statement: The study was commissioned by the Hessian Ministry of Social Affairs and Integration and was supported by Roche, Basel, Switzerland.Declaration of Interests: None to declare.Ethics Approval Statement: This study protocol was approved by the ethics board of the University Hospital Frankfurt, Goethe University Frankfurt am Main, Germany.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings (preprint)

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. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate, SDS, and Triton-X containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In addition, the stability of the virus in cell culture media at 4{degrees}C and on surfaces used in laboratory environment was analyzed. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.