Linear epitope-specific IgG responses to SARS-CoV-2 and related human coronaviruses in human COVID-19 convalescent sera
ABSTRACT: The on-going COVID-19 pandemic requires a deeper understanding of the long-term antibody responses that persist following SARS-CoV-2 infection. To that end, we determined epitope-specific IgG antibody responses in COVID-19 convalescent sera collected at 5 months post-diagnosis and compared that to sera from naïve individuals. Each serum sample was reacted with a high-density peptide microarray representing the complete proteome of SARS-CoV-2 as 15 mer peptides with 11 amino acid overlap and homologs of spike glycoprotein, nucleoprotein, membrane protein, and envelope small membrane protein from related human coronaviruses. Binding signatures were compared between COVID-19 convalescent patients and naïve individuals using the web service tool EPIphany.
Project description:ABSTRACT Pandemic SARS-CoV-2 has caused unprecedented mortalities. Vaccine is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with the elicited neutralizing antibodies in convalescent population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent sera and 24 non-COVID-19 sera. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent sera. Of note, two peptides non-specifically interacted with most of the non-COVID-19 sera. Neutralization assay indicated that only five sera completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent sera, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.
Project description:Coronaviruses share conservative spike protein (S) on their enveloped membrane surface, where S1 subunit recognizes and binds the cellular receptor, and the S2 subunit mediates membrane fusion. This similarity raises the question: does coronaviral infection by one create protection to others? Convalescent SARS-CoV-2 (COVID-19) sera were tested for cross reactivity with peptides from Middle East respiratory syndrome coronavirus (MERS-CoV) which shares 74% homology. Our results showed significant cross-reactivity with a peptide of the heptad repeat 2 (HR2) domain of the MERS-CoV spike protein. Sera samples of 47 validated seropositive convalescent COVID-19 patients and 40 sera samples of control patients, collected in pre-COVID time were used to establish cross-bind reactivity with the MERS-CoV peptide. Significantly stronger binding (p?<?0.0001) was observed for IgG antibodies in convalescent COVID-19 patients compared to the control group. In ELISA, MERS-CoV peptide helps to discriminate post-COVID-19 populations and non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection can recognize and probably decrease severity of MERS-CoV and other coronaviral infections. The high homology of the spike protein domain also suggests that the opposite effect can be true: coronaviral infections produce cross-reactive antibodies effective against SARS-CoV-2. The collected data prove that despite the core HR2 region is hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections and developing vaccines effective even after possible viral mutations.
Project description:Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was ~93% (PRNT50) and ~54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 ELISA titers) showed maximal activity, but not all high titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.
Project description:We utilize single-cell sequencing (scSeq) of lymphocyte immune repertoires and transcriptomes to quantitatively profile the adaptive immune response in COVID-19 patients of varying age. Our scSeq analysis defines the adaptive immune repertoire and transcriptome in convalescent COVID-19 patients and shows important age-related differences implicated in immunity against SARS-CoV-2.
Project description:Replication-competent vesicular stomatitis virus (VSV)-based recombinant viruses are useful tools for studying emerging and highly pathogenic enveloped viruses in level 2 biosafety facilities. Here, we used a replication-competent recombinant VSVs (rVSVs) encoding the spike (S) protein of SARS-CoV-2 in place of the original G glycoprotein (rVSV-eGFP-SARS-CoV-2) to develop a high-throughput entry assay for SARS-CoV-2. The S protein was incorporated into the recovered rVSV-eGFP-SARS-CoV-2 particles, which could be neutralized by sera from convalescent COVID-19 patients. The recombinant SARS-CoV-2 also displayed entry characteristics similar to the wild type virus, such as cell tropism and pH-dependence. The neutralizing titers of antibodies and sera measured by rVSV-eGFP-SARS-CoV-2 were highly correlated with those measured by wild-type viruses or pseudoviruses. Therefore, this is a safe and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines and therapeutics.
Project description:There is an urgent need for effective treatment and preventive vaccine to contain this devastating global pandemic, which requires a comprehensive understanding of humoral responses specific to SARS-CoV-2 during the disease progression and convalescent phase of COVID-19 patients. We continuously monitored the serum IgM and IgG responses specific to four SARS-CoV-2 related antigens, including the nucleoprotein (NP), receptor binding domain (RBD), S1 protein, and ectodomain (ECD) of the spike protein among non-severe and severe COVID-19 patients for seven weeks since disease onset. Most patients generated humoral responses against NP and spike protein-related antigens but with their distinct kinetics profiles. Combined detection of NP and ECD antigens as detecting antigen synergistically improved the sensitivity of the serological assay, compared to that of using NP or RBD as detection antigen. 80.7% of convalescent sera from COVID-19 patients revealed that the varying extents of neutralization activities against SARS-CoV-2. S1-specific and ECD-specific IgA responses were strongly correlated with the neutralization activities in non-severe patients, but not in severe patients. Moreover, the neutralizing activities of the convalescent sera were shown to significantly decline during the period between 21 days to 28 days after hospital discharge, accompanied by a substantial drop in RBD-specific IgA response. Our data provide evidence that are crucial for serological testing, antibody-based intervention, and vaccine design of COVID-19.
Project description:To elucidate the T cell epitopes of SARS-CoV-2, we stimulated human PBMCs from healthy donors and convalescent COVID-19 patients with various SARS-CoV-2 antigens, sorted the activated T cells and performed sc-RNA and -TCR sequencing. We obtained thousands of T cell clonotypes that responded to SARS-CoV-2 antigens, and identified the epitopes and restricting HLAs of several clonotypes that were significantly expanded in the COVID-19 patients. Overall design: Determination of the TCR and mRNA expression of human T cells stimulated with SARS-CoV-2 antigens.
Project description:Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and quickly spread globally. In this study, we investigated the characteristics of viral shedding from different sites and the neutralizing antibody (NAb) response during the acute and convalescent phases of nine children with COVID-19. SARS-CoV-2 was detected in their nasopharyngeal swabs (9/9, 100%), stool samples (8/9, 89%), and oropharyngeal swabs (3/9, 33%) but was not detected in their serum and urine samples. The median duration of viral shedding detected in nasopharyngeal swabs, oropharyngeal swabs, and stools was 13, 4, and 43 days respectively, and the maximum duration of viral shedding detected from stools was 46 days after discharge. In children, nasopharyngeal swabs appear to be a more sensitive specimen type for the diagnosis of COVID-19 compared with oropharyngeal swabs. Three of eight patients produced NAbs in the acute phase, and NAbs were detected in all eight patients with convalescent sera. The results of this study provide valuable information for the diagnosis and surveillance of COVID-19 and development of SARS-CoV-2 vaccines for use in children.
Project description:The recently emerged coronavirus SARS-CoV-2, the causative agent of COVID-19, is rapidly spreading in the world. The exponentially expanding threat of SARS-CoV-2 to global health highlights the urgent need for a vaccine. Herein we show the rapid development of a novel, highly efficient, and safe COVID-19 vaccine using a rabies virus-based vector that has proven to be an efficient vaccine against several emerging infectious diseases. This study reports that both a live and an inactivated rabies virus containing the SARS-CoV-2 spike S1 protein induces potent virus-neutralizing antibodies at much higher levels than seen in the sera of convalescent patients. In summary, the results provided here warrant further development of this safe and established vaccine platform against COVID-19.
Project description:The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates complete discrimination of these two groups. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.