Discriminating promiscuous from target-specific autoantibodies in COVID-19.

Diverse autoantibodies were suggested to contribute to severe outcomes of COVID-19, but their functional implications are largely unclear. ACE2, the SARS-CoV-2 receptor and a key regulator of blood pressure, was described to be one of many targets of autoantibodies in COVID-19. ACE2 in its soluble form (sACE2) is highly elevated in the blood of critically ill patients, raising the question of whether sACE2:spike complexes induce ACE2 reactivity. Screening 247 COVID-19 patients, we observed elevated sACE2 and anti-ACE2 IgG that were poorly correlated. Interestingly, levels of IgGs recognizing ACE2, IFNα2, and CD26 strongly correlated in severe COVID-19, with 15% of sera showing polyreactivity versus 4.1% exhibiting target-directed autoimmunity. Promiscuous autoantibodies failed to impair the activity of ACE2 and IFNα2, while only specific anti-IFNα2 IgG compromised cytokine function. Our study suggests that the detection of autoantibodies in COVID-19 is often attributed to a promiscuous reactivity, potentially misinterpreted as target-specific autoimmunity with functional impact.

A design strategy to generate a SARS-CoV-2 RBD vaccine that abrogates ACE2 binding and improves neutralizing antibody responses.

The structure-based design of antigens holds promise for developing vaccines with higher efficacy and improved safety profiles. We postulate that abrogation of host receptor interaction bears potential for the improvement of vaccines by preventing antigen-induced modification of receptor function as well as the displacement or masking of the immunogen. Antigen modifications may yet destroy epitopes crucial for antibody neutralization. Here, we present a methodology that integrates deep mutational scans to identify and score SARS-CoV-2 receptor binding domain variants that maintain immunogenicity, but lack interaction with the widely expressed host receptor. Single point mutations were scored in silico, validated in vitro, and applied in vivo. Our top-scoring variant receptor binding domain-G502E prevented spike-induced cell-to-cell fusion, receptor internalization, and improved neutralizing antibody responses by 3.3-fold in rabbit immunizations. We name our strategy BIBAX for body-inert, B-cell-activating vaccines, which in the future may be applied beyond SARS-CoV-2 for the improvement of vaccines by design.

Soluble ACE2 correlates with severe COVID-19 and can impair antibody responses.

Identifying immune modulators that impact neutralizing antibody responses against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is of great relevance. We postulated that high serum concentrations of soluble angiotensin-converting enzyme 2 (sACE2) might mask the spike and interfere with antibody maturation toward the SARS-CoV-2-receptor-binding motif (RBM). We tested 717 longitudinal samples from 295 COVID-19 patients and showed a 2- to 10-fold increase of enzymatically active sACE2 (a-sACE2), with up to 1 µg/mL total sACE2 in moderate and severe patients. Fifty percent of COVID-19 sera inhibited ACE2 activity, in contrast to 1.3% of healthy donors and 4% of non-COVID-19 pneumonia patients. A mild inverse correlation of a-sACE2 with RBM-directed serum antibodies was observed. In silico, we show that sACE2 concentrations measured in COVID-19 sera can disrupt germinal center formation and inhibit timely production of high-affinity antibodies. We suggest that sACE2 is a biomarker for COVID-19 and that soluble receptors may contribute to immune suppression informing vaccine design.

Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.