Project description:Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.
Project description:Serological assays have been widely used to detect anti-SARS-CoV-2 antibodies, which are generated from previous exposure to the virus or after vaccination. The presence of anti-SARS-CoV-2 Nucleocapsid antibodies was recently reported in patients´ urine using an in-house urine-based ELISA-platform, allowing a non-invasive way to collect clinical samples and assess immune conversion. In the current study, we evaluated and validated another in-house urine-based ELISA for the detection of anti-SARS-CoV-2 Spike antibodies. Three partial recombinant SARS-CoV-2 Spike proteins comprising the Receptor Binding Domain, expressed in eukaryotic or prokaryotic systems, were tested in an ELISA platform against a panel of over 140 urine and paired serum samples collected from 106 patients confirmed positive for SARS-CoV-2 by qRT-PCR. The key findings from our study were that anti-SARS-CoV-2 Spike antibodies could be detected in urine samples and that the prokaryotic expression of the rSARS-CoV-2 Spike protein was not a barrier to obtain relatively high serology efficiency for the urine-based assay. Thus, use of a urine-based ELISA assay with partial rSARS-CoV-2 Spike proteins, expressed in a prokaryotic system, could be considered as a convenient tool for screening for the presence of anti-SARS-CoV-2 Spike antibodies, and overcome the difficulties arising from sample collection and the need for recombinant proteins produced with eukaryotic expression systems.
Project description:SARS-CoV-2 causing the worldwide pandemic has changed people's life in multiple aspects dramatically since it's first identified in Wuhan, China at the end of 2019. While the numbers of infected patients and death toll keep vigorous increasing, curbing the progression of the pandemic is an urgent goal. Efforts have been made to search for prophylactic and therapeutic approaches including neutralizing antibodies development. By reviewing dozens of studies on anti-spike antibodies identification, we concluded that (1) promising therapeutic antibodies are being fished out by various approaches, such as screening of single B cells of convalescent patients, recombinant antibody library and B cells of immunized animals; (2) the epitopes are mainly RBD, but also some non-RBD domains, without the requisite of overlapping with ACE2 binding sites; (3) Neutralizing antibodies are convergent to a few germline genes, including IGHV3-30, IGHV3-53, IGHV3-66, with varying levels of somatic mutations. This review summarizes the progress in neutralizing antibodies development and the germline enrichment of effective antibodies, which will shed light on COVID-19 treatment and vaccine design.
Project description:Dysregulated immune responses contribute to the excessive and uncontrolled inflammation observed in severe COVID-19. However, how immunity to SARS-CoV-2 is induced and regulated remains unclear. Here we uncover a role of the complement system in the induction of innate and adaptive immunity to SARS-CoV-2. Complement rapidly opsonizes SARS-CoV-2 particles via the lectin pathway. Complement-opsonized SARS-CoV-2 efficiently induces type-I interferon and pro-inflammatory cytokine responses via activation of dendritic cells, which are inhibited by antibodies against the complement receptors (CR) 3 and 4. Serum from COVID-19 patients, or monoclonal antibodies against SARS-CoV-2, attenuate innate and adaptive immunity induced by complement-opsonized SARS-CoV-2. Blocking of CD32, the FcγRII antibody receptor of dendritic cells, restores complement-induced immunity. These results suggest that opsonization of SARS-CoV-2 by complement is involved in the induction of innate and adaptive immunity to SARS-CoV-2 in the acute phase of infection. Subsequent antibody responses limit inflammation and restore immune homeostasis. These findings suggest that dysregulation of the complement system and FcγRII signaling may contribute to severe COVID-19.
Project description:Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.
Project description:Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.
Project description:A better understanding of the anti-SARS-CoV-2 immune response is necessary to finely evaluate commercial serological assays but also to predict protection against reinfection and to help the development of vaccines. For this reason, we monitored the anti-SARS-CoV-2 antibody response in infected patients. In order to assess the time of seroconversion, we used 151 samples from 30 COVID-19 inpatients and monitored the detection kinetics of anti-S1, anti-S2, anti-RBD and anti-N antibodies with in-house ELISAs. We observed that specific antibodies were detectable in all inpatients 2 weeks post-symptom onset and that the detection of the SARS-CoV-2 Nucleocapsid and RBD was more sensitive than the detection of the S1 or S2 subunits. Using retroviral particles pseudotyped with the spike of the SARS-CoV-2, we also monitored the presence of neutralizing antibodies in these samples as well as 25 samples from asymptomatic individuals that were shown SARS-CoV-2 seropositive using commercial serological tests. Neutralizing antibodies reached a plateau 2 weeks post-symptom onset and then declined in the majority of inpatients but they were undetectable in 56% of asymptomatic patients. Our results indicate that the SARS-CoV-2 does not induce a prolonged neutralizing antibody response. They also suggest that induction of neutralizing antibodies is not the only strategy to adopt for the development of a vaccine. Finally, they imply that anti-SARS-CoV-2 neutralizing antibodies should be titrated to optimize convalescent plasma therapy.
Project description:While there is SARS-CoV-2 multiorgan tropism in severely infected COVID-19 patients, it's unclear if this occurs in healthy young individuals. In addition, for antibodies that target the spike protein (SP), it's unclear if these reduce SARS-CoV-2/SP multiorgan tropism equally. We used fluorescently labeled SP-NIRF to study viral behavior, using an in vivo dynamic imaging system and ex in vivo tissue analysis, in young mice. We found a SP body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. SP uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or CSF. Thus, the brain vascular barriers were effective in restricting the entry of SP into brain parenchyma in young healthy mice. While both anti-ACE2 and anti-SP antibodies suppressed SP biodistribution and organ uptake, anti-SP antibody was more effective. By extension, our data support the efficacy of these antibodies on SARS-CoV-2 multiorgan tropism, which could determine COVID-19 organ-specific outcomes.