SARS-CoV-2 spike (S) variants govern transmissibility, responsiveness to vaccination and disease severity. In a screen for new models of SARS-CoV-2 infection, we identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2 expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Though the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.
Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.
Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR Green assay was developed for T. brucei in a 384-well format. This semi-automated assay is cost- and time-effective, robust, and reproducible. The SYBR Green assay was compared to the resazurin assay by screening a library of 4000 putative kinase inhibitors, revealing a superior performance in terms of assay time, sensitivity, simplicity, and reproducibility, and resulting in a higher hit confirmation rate. Although the resazurin assay allows for comparatively improved detection of slow-killing compounds, it also has higher false-positive rates that are likely to arise from the assay experimental conditions. The compounds with the most potent antitrypanosomal activity were selected in both screens and grouped into 13 structural clusters, with 11 new scaffolds as antitrypanosomal agents. Several of the identified compounds had IC50 <1 µM coupled with high selectivity toward the parasite. The core structures of the scaffolds are shown, providing promising new starting points for drug discovery for HAT.
Background There are limited data directly comparing immune responses to vaccines and to natural infections with COVID-19. This study assessed the immunogenicity of the BNT162b2 and ChAdOx1 nCoV-19 vaccines over a 3-month period and compared the immune responses with those to natural infections. Method We enrolled healthcare workers (HCWs) who received BNT162b2 or ChAdOx1 nCoV-19 vaccines and COVID-19-confirmed patients, and then S1-IgG and neutralizing antibodies and T cell responses were measured. Results A total of 121 vaccinees and 26 patients with confirmed COVID-19 were analyzed. After the 2 nd dose, the BNT162b2 vaccine yielded S1-IgG antibody responses similar to natural infections (2241 ± 899 vs. 2601 ± 5039, p=0.676), but significantly stronger than the ChAdOx1 vaccine (174 ± 96, p <0.0001). The neutralizing antibody titer generated by BNT162b2 was 6-fold higher than that generated by ChAdOx1, but lower than that by natural infection. T cell responses persisted for the 3 months in the BNT162b2 and natural infection but decreased in the ChAdOx1. Conclusions Antibody responses after the 2 nd dose of BNT162b2 are higher than after the 2 nd dose of ChAdOx1 and like those occurring after natural infection. T cell responses are maintained longer in BNT162b2 vaccinees than in ChAdOx1 vaccinees.
In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a β-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50 values from two assays showed a very poor correlation, with an R2 value of 0.005. Nitroheterocyclics and CYP51 inhibitors were inactive in an image-based assay, but were highly active in a colorimetric assay. In order to identify the differentiating factor, we synchronized the compound–parasite incubation times or the sequential cell and compound seeding schemes between assays, but the correlation remained low. A high correlation (R2 = 0.86) was observed only after both compound incubation time and cell seeding were synchronized between assays. Further analysis of EC50 and maximum inhibition values showed that nitroheterocyclics and CYP51 inhibitors exhibit relatively large deviations in activity between experimental protocols routinely used for in vitro intracellular T. cruzi assays. These findings suggest that the factors mentioned are critical when designing an intracellular T. cruzi assay.
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