Project description:This experiment aims to profile polyclonal antibody binding profiles in serum from vaccinated animals relative to antibody function in a virus neutralization assay. Rabbits received three vaccinations with a DNA vaccine encoding the spike protein of the SARS-CoV-2 index strain. Serum samples were selected based on a three-tier (low, intermediate, and high) capacity to cross-neutralize SARS-CoV-2 strains with known neutralization resistance. Following normalization of total anti-spike IgG levels, serum of each animal (n=3) were evaluated for antibody binding to 10mer cyclic constrained peptides spanning the entire spike protein and regions with known SARS-CoV-2 variant of concern spike mutations.
Project description:We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal (HCoV-NL63, -229E, -OC43 and -HKU1) and epidemic coronaviruses (SARS-CoV, hCoV-MERS) at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed IgG antibody reactivity to nucleocapsid and spike antigens using protein microarray. A cutoff was set at the average plus 3 times the SD of 20 nonreactive cultures with a minimum MFI of 1000.
Project description:Single-cell RNA sequencing (scRNA-seq) has aided greatly in the study of viruses to distinguish responses from infected versus bystander cells in complex systems. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories. Here we show that TCL buffer (Qiagen), inactivates both Ebola virus (EBOV) and SARS-CoV-2, representative BSL-4 and BSL-3 viruses. We show that additional heat treatment was additionally sufficient to inactivate EBOV-containing samples, and had minimal effects on extracted RNA quality and downstream sequencing results.
Project description:We performed genome-wide CRISPR KO screens in human Huh7.5.1 cells to select for mutations that render host cells resistant to viral infection by SARS-CoV-2, human coronavirus 229E and OC43.
Project description:Coronaviruses express a repertoire of accessory proteins for evading host immune responses. Among these accessory proteins, a small internal (I) protein is expressed by members of the genus Betacoronavirus. Previous studies reported that the I proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 inhibit type I interferon (IFN-I) expression through distinct mechanisms and play different roles in pathogenesis. In addition, human coronaviruses HKU1 and OC43 are betacroaonvairuses that predominantly cause common cold and encode the I protein as one of their accessory proteins; the I proteins of hCoV-HKU1 and hCoV-OC43 have not been previously characterized. However, the lack of robust reverse genetic systems, tissue culture and animal models limit the study of hCoV-HKU1 and hCoV-OC43 pathogenesis. Here, we examined the role of hCoV-HKU1 and hCoV-OC43 I proteins in pathogenesis using a prototypic coronavirus. We introduced the I proteins of hCoV-HKU1 and hCoV-OC43 independently to a neurotropic strain of mouse hepatitis virus (MHV-J2.2). MHV-J2.2 infection is well-characterized with clearly defined immune responses which allows the study of I proteins in the context of authentic coronavirus infection. We showed that the I protein of hCoV-HKU1 but not that of hCoV-OC43 ameliorated MHV-J2.2 infection while the I protein of MERS-CoV causes exacerbated disease. Further analysis revealed that infection with MHV-J2.2 expressing the I protein of MERS-CoV leads to increased neutrophil infiltration to the site of infection and virus titers in mice; diminished virus titers was observed in the presence of hCoV-HKU1 I protein. Overall, our findings suggest that the I protein of different betacoronaviruses play unique roles in pathogenesis.
Project description:Despite the clinical success of anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines by rapidly spreading SARS-CoV-2 variants has been compromised. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host’s immune response and attenuate antibody efficiency. However, it still remains unclear whether targeting glycosylation on spike can impair SARS-CoV-2 and its variants infectivity. Methods: To assess the binding ability of glycosylated or deglycosylated spike with ACE2, we performed flow cytometry, ELISA, and BioLayer Interferometry methods. Viral entry ability was determined by luciferase intensity, immunoblotting, and immunofluorescence assay. A genome-wide association study (GWAS) was performed to identify the relationship of STT3A and COVID-19 severity. N-glycosylation regulated by NF-kB/STT3A axis was investigated by knockdown approach, chromatin immunoprecipitation, and promoter assay. To specifically target SARS-CoV-2 infected cells, we developed an antibody-drug conjugate coupling non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) on inhibitory effects of SARS-CoV-2 infection. Results: We found receptor binding domain and three SARS-CoV-2 distinct surface Nglycosylation sites in 57,311 spikes retrieved from NCBI-Virus-database are highly evolutionarily conserved (99.67%) and involved in ACE2 interaction. We further identified STT3A as a key glycosyltransferase that catalyzed spike glycosylation and positively correlated with COVID-19 severity. Inhibition of STT3A by N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 and its variants (B.1.1.7, and B.1.351) infectivity. Most importantly, 4G10-ADC internalized SARS-CoV-2 infected cells and subsequently released NGI-1 to deglycosylate spike protein. Thereby, it reinforces the neutralizing abilities in antibodies, vaccines, or convalescent sera, inhibiting SARS-CoV-2 and its variants’ infectivity. Our results suggest targeting STT3A-mediated evolution conserved glycosylation via ADC can provide a widespread impact on SARS-CoV-2 variants infection. Together, we identified a novel deglycosylation method to eradicate SARS-CoV-2 variants infection.
Project description:The viral RNA-dependent RNA polymerase (replicase) from Venezuelan equine encephalitis virus constitutes a vital component of the bipartite trans-amplifying mRNA vaccine. In this vaccine strategy aimed at targeting SARS-CoV-2, the replicase mRNA is administered alongside the mRNA encoding the SARS-CoV-2 spike protein. Our investigation sought to determine whether the replicase induces amplification of cellular mRNAs. To this end, cells were transfected with mRNAs encoding the replicase and SARS-CoV-2 spike protein, while control groups received transfections of mRNAs encoding an unrelated protein along with the SARS-CoV-2 spike. We observed no significant upregulation of genes in the treatment group compared to the control group. This suggests that the replicase does not induce off-target amplification of cellular mRNAs.
Project description:Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. Vaccine was dosed to induce binding antibody titers against ancestral spike, but not efficient virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post infection reflects both vaccination status and disease course, and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of neutralizing antibodies, correlates with recall of broadly reactive B- and T-cell responses.
Project description:Recent exposure to seasonal coronaviruses (sCoVs) may stimulate cross-reactive antibody responses against SARS-CoV-2. Previous studies have shown divergent results regarding protective or damaging immunity induced by prior exposure to sCoVs. It is still unknown whether pre-existing humoral immunity may play a role in the vaccine-induced neutralization and antibody responses. In this study, we collected 36 paired sera in healthy volunteers before and after immunization with inactivated SARS-CoV-2 vaccines, and analyzed the distribution and intensity of pre-existing antibody responses at the epitope level before vaccine immunization, as well as the relationship between pre-existing sCoVs immunity and vaccine-induced neutralization.
Project description:Recent exposure to seasonal coronaviruses (sCoVs) may stimulate cross-reactive antibody responses against SARS-CoV-2. Previous studies have shown divergent results regarding protective or damaging immunity induced by prior exposure to sCoVs. It is still unknown whether pre-existing humoral immunity may play a role in the vaccine-induced neutralization and antibody responses. In this study, we collected 36 paired sera in healthy volunteers before and after immunization with inactivated SARS-CoV-2 vaccines, and analyzed the distribution and intensity of pre-existing antibody responses at the epitope level before vaccine immunization, as well as the relationship between pre-existing sCoVs immunity and vaccine-induced neutralization.