Antibody binding and ACE2 binding inhibition is significantly reduced for the Omicron variant compared to all other variants of concern

The rapid emergence of the Omicron variant and its large number of mutations has led to its classification as a variant of concern (VOC) by the WHO(1). Initial studies on the neutralizing response towards this variant within convalescent and vaccinated individuals have identified substantial reductions(2-8). However many of these sample sets used in these studies were either small, uniform in nature, or were compared only to wild-type (WT) or, at most, a few other VOC. Here, we assessed IgG binding, (Angiotensin-Converting Enzyme 2) ACE2 binding inhibition, and antibody binding dynamics for the omicron variant compared to all other VOC and variants of interest (VOI)(9), in a large cohort of infected, vaccinated, and infected and then vaccinated individuals. While omicron was capable of binding to ACE2 efficiently, antibodies elicited by infection or immunization showed reduced IgG binding and ACE2 binding inhibition compared to WT and all VOC. Among vaccinated samples, antibody binding responses towards omicron were only improved following administration of a third dose. Overall, our results identify that omicron can still bind ACE2 while pre-existing antibodies can bind omicron. The extent of the mutations appear to inhibit the development of a neutralizing response, and as a result, omicron remains capable of evading immune control.

Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

The SARS-CoV-2 pandemic virus is consistently evolving with mutations within the receptor binding domain (RBD)1 being of particular concern2-4. To date, there is little research into protection offered following vaccination or infection against RBD mutants in emerging variants of concern (UK3, South African5, Mink6 and Southern California7). To investigate this, serum and saliva samples were obtained from groups of vaccinated (Pfizer BNT-162b28), infected and uninfected individuals. Antibody responses among groups, including salivary antibody response and antibody binding to RBD mutant strains were examined. The neutralization capacity of the antibody response against a patient-isolated South African variant was tested by viral neutralization tests and further verified by an ACE2 competition assay. We found that humoral responses in vaccinated individuals showed a robust response after the second dose. Interestingly, IgG antibodies were detected in large titers in the saliva of vaccinated subjects. Antibody responses showed considerable differences in binding to RBD mutants in emerging variants of concern. A substantial reduction in RBD binding and neutralization was detected for the South African variant. Taken together our data reinforces the importance of administering the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies. High antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant highlights importance of surveillance strategies to detect new variants and targeting these in future vaccines.

cAMP prevents antibody-mediated thrombus formation in COVID-19

Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. However, the exact mechanisms of thromboembolic events remain elusive. In this work, we show that immunoglobulin G (IgG) subclass in patients with COVID-19 trigger the formation of procoagulant PLTs in a Fc-gamma-RIIA (Fc{gamma}RIIA) dependent pathway leading to increased thrombus formation in vitro. Most importantly, these events were significantly inhibited via Fc{gamma}RIIA blockade as well as by the elevation of PLTs intracellular cyclic-adenosine-monophosphate (cAMP) levels by the clinical used agent Iloprost. The novel findings of Fc{gamma}RIIA mediated prothrombotic conditions in terms of procoagulant PLTs leading to higher thrombus formation as well as the successful inhibition of these events via Iloprost could be promising for the future treatment of the complex coagulopathy observed in COVID-19 disease. Key points- Fc-gamma-receptor IIA mediated PS externalization on the PLT surface triggers increased thrombus formation - Inductors of cAMP inhibit antibody-mediated thrombus formation and may have potential therapeutic advantage in COVID-19

Severe COVID-19 infection is associated with increased antibody-mediated platelet apoptosis

The pathophysiology of COVID-19 associated thrombosis seems to be multifactorial, involving interplay between cellular and plasmatic elements of the hemostasis. We hypothesized that COVID-19 is accompanied by platelet apoptosis with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential ({Delta}{Psi}m), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization by flow cytometry. Platelets from intensive care unit (ICU) COVID-19 patients (n=21) showed higher {Delta}{Psi}m depolarization, cytosolic Ca2+ concentration and PS externalization, compared to healthy controls (n=18) and COVID-19 non-ICU patients (n=4). Moreover significant higher cytosolic Ca2+ concentration and PS was observed compared to septic ICU control group (ICU control). In ICU control group (n=5; ICU non-COVID-19) cytosolic Ca2+ concentration and PS externalization was comparable to healthy control, with an increase {Delta}{Psi}m depolarization. Sera from ICU COVID-19 13 patients induced significant increase in apoptosis markers ({Delta}{Psi}m depolarization, cytosolic Ca2+ concentration and PS externalization). compared to healthy volunteer and septic ICU control. Interestingly, immunoglobulin G (IgG) fractions from COVID-19 patients induced an Fc gamma receptor IIA dependent platelet apoptosis ({Delta}{Psi}m depolarization, cytosolic Ca2+ concentration and PS externalization). Enhanced PS externalization in platelets from ICU COVID-19 patients was associated with increased sequential organ failure assessment (SOFA) score (r=0.5635) and DDimer (r=0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared to those without. The strong correlations between apoptosis markers and increased D-Dimer levels as well as the incidence of thrombosis may indicate that antibody-mediated platelet apoptosis potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients. Key pointsO_LISevere COVID-19 is associated with increased antibody-mediated platelet apoptosis. C_LIO_LIPlatelet apoptosis in severe COVID-19 is correlated with D-Dimer and higher incidence of thromboembolisms. C_LI

Neutralizing antibody response in non-hospitalized SARS-CoV-2 patients

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2 and two other patients (4%) were only positive in one of the six serological assays employed. For the remainder, antibody response against the S-protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. Regarding neutralization, only six patients (12%) could be classified as highly neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.

Going beyond clinical routine in SARS-CoV-2 antibody testing - A multiplex corona virus antibody test for the evaluation of cross-reactivity to endemic coronavirus antigens

Given the importance of the humoral immune response to SARS-CoV-2 as a global benchmark for immunity, a detailed analysis is needed to monitor seroconversion in the general population, understand manifestation and progression of COVID-19 disease, and ultimately predict the outcome of vaccine development. In contrast to currently available serological assays, which are only able to resolve the SARS-CoV-2 antibody response on an individual antigen level, we developed a multiplex immunoassay, for which we included spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses (NL63, OC43, 229E, HKU1) in an expanded antigen panel. Compared to three commercial in vitro diagnostic tests, our MULTICOV-AB assay achieved the highest sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. Simultaneously, high IgG responses against endemic coronaviruses became apparent throughout all samples, but no consistent cross-reactive IgG response patterns could be defined. In summary, we have established and validated, a robust, high-content-enabled, and antigen-saving multiplex assay MULTICOV-AB, which is highly suited to monitor vaccination studies and will facilitate epidemiologic screenings for the humoral immunity toward pandemic as well as endemic coronaviruses.