Kinetic analysis of de novo autoreactivity in severe COVID-19 reveals important implications for early intervention and treatment in patients with post-COVID sequelae.
The SARS-CoV-2 outbreak was first declared a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30, 2020, and upgraded to pandemic status just five weeks later.1 This instigated an unprecedented mobilization of resources and research efforts towards treating and preventing COVID-19 infection. Elucidating underlying mechanisms of disease pathogenesis became imperative for disease stratification, devising diagnostics tools, and developing effective treatments and vaccines to contain the pandemic and improve patient outcomes.
Early characterization of severe COVID-19 disease suggested immune responses were not only responsible for viral clearance, but possibly contributed to disease pathology.2-5 This was corroborated by clinical manifestations in COVID-19 patients similar to autoimmune features6,7, including the presence of autoreactive antibodies.8
To date, there have been over 633 million confirmed cases of COVID-19 worldwide, including 6.6 million deaths.9 While infection and mortality rates have since fallen, experts agree COVID isn’t going anywhere10 and continued research is key in keeping up with the evolution of this virus. A recent publication by Woodruff et al. in Nature further investigated the presence and role of de novo autoreactive antibodies in COVID-19 and their implication in disease pathogenesis.11
A key component of this study was the use of Alto, the world’s first digital SPR instrument, to characterize the binding kinetics of monoclonal antibodies with identified autoreactivity to SARS-CoV-2 viral proteins. Below, we summarize the study’s findings and Alto’s role in the researchers’ workflow.
Table of contents
- De novo autoreactivity in severe COVID-19
- Antibody-secreting cells (ASCs) linked to de novo autoreactivity
- Assessing COVID-19 patient samples for autoreactivity
- Characterizing binding kinetics of select mAbs using Surface Plasmon Resonance (SPR)
- Evaluating cross-reactivity of antibodies between self-antigens and the RBD
De novo autoreactivity in severe COVID-19
Deep analysis of B cell activation pathways by Ignacio Sanz’ group12 has led to a strong emphasis on the extrafollicular (EF) pathway — a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity13 — as a common feature of severe COVID-19 disease.
To further investigate the developmental origins of these autoreactive antibodies and their connection with the intrinsic antiviral response, a research study was carried out by Matthew C. Woodruff et al.11 The team’s findings presented in their recent Nature publication, Dysregulated naive B cells and de novo autoreactivity in severe COVID-19, shine a light on the origins, breadth, and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.
Antibody-secreting cells (ASCs) linked to de novo autoreactivity
First, the researchers confirmed the relevance of early circulating antibody-secreting cells (ASCs) to the antiviral response, using a new in vitro method that optimizes overnight antibody secretion from ASCs into the culture supernatant. Next, single-cell VDJ repertoire analysis was performed to study the nature of the ASC compartment in patients. Isotype analysis demonstrated a consistent expansion of IgG1 in the intensive care unit (ICU) cohort. The cohort’s expanded IgG1+ ASC compartment was characterized by reduced mutation frequency, largely concentrated on the IgG1 compartment.
To better understand the origins and persistence of the low-mutation IgG1 ASC compartment, CD27+ memory B cells were sorted and analyzed. The findings indicated uncoupling and separate selection pressures between the IgG1 ASC and memory B cell repertoires, consistent with the emergence during acute severe infection of a memory-independent, newly generated ASC compartment with reduced selective pressure.
Assessing COVID-19 patient samples for autoreactivity
To elucidate whether COVID-19 responses also correlated with autoreactivity, plasma samples collected from 79 individuals were tested for more than 30 clinically relevant autoantigens, and later analyzed for autoreactivity associated with connective tissue disorders. Higher ‘densities’ of autoreactivity were significantly increased in ICU patients, while autoreactivity screening identified the emergence of two autoreactivities: antinuclear antibodies (ANAs) and anti-carbamylated protein responses (CarP). Anti-CarP antibodies were specific to the ICU cohort and present in more than 40% of patients.
To characterize specificity to COVID-19, 28 plasma samples from ICU patients with acute respiratory distress syndrome were assessed for autoreactivity. Remarkably, the autoreactivity profiles of these patients were highly similar to those of patients with critical COVID-19, suggesting the autoimmune phenomena described in COVID-19 may be generalizable to other severe pulmonary infections.
Characterizing binding kinetics of select mAbs using Surface Plasmon Resonance (SPR)
Next, two ICU patients — representative of the overall cohort — were identified for individual clonotype assessment and monoclonal antibody (mAb) production and testing.
A pool of 54 mAbs from the two selected patients was first prescreened for antigen binding through Luminex-based multiplex binding assessment. Subsequently, five select antibodies were fully characterized using Alto’s single-cycle kinetics (SCK) analysis against either the SARS-CoV-2 nucleocapsid or spike trimer protein as the ligand. All data were collected in real time on a 16-channel carboxyl-coated sensor cartridge with a 1:1 referencing format. Alto automated all sample dilutions, generating five threefold analyte dilutions for each SCK run, with final concentrations between 222 nM and 914 pM.
The kinetic data revealed that the naive-derived, low-mutation monoclonal antibodies exhibited high affinity for the spike trimer and nucleocapsid, with KD values in the low nanomolar range. The top binders to spike trimer and nucleocapsid had affinities of 2.82 nM and 0. 993 nM, respectively, in the range of published neutralizing antibodies.14
More than 65% of mAbs showed binding to one of the tested target antigens. Despite their naive origin, many of the resulting antibodies had high affinity as determined by SPR, confirming the contribution of the IgG1 compartment enriched for antigen-specific ASCs to the emerging antiviral response.
The use of Alto’s high-throughput digital SPR platform enabled simultaneous, real-time analysis of multiple samples in a single cartridge, while automating all serial dilutions and reducing sample requirements to a fraction of traditional SPR systems. The efficient elucidation of kinetics data was pivotal to the granularity of the study’s results and interpretations.
Evaluating cross-reactivity of antibodies between self-antigens and the RBD
Despite the dominance of SARS-CoV-2-specific ASCs, 30% of the clones tested either did not have clear specificity for the tested SARS-CoV-2 proteins, or showed low binding. To better understand whether these antibodies contained autoreactive potential, mAbs were screened for ANA binding, with 16% of all mAbs showing ANA reactivity. This demonstrated the potential of common, clinically testable autoreactivity tests — including ANAs and CarP reactivity — in identifying these phenomena in a variety of severe infectious diseases in real time.
In total, 65% (15/23) of mAbs with identified autoreactivity had some affinity to a screened viral antigen. Cross-reactivity between self-antigens and the RBD confirmed the naive origins of the autoreactive response. The heterogeneity of antiviral targets associated with self-reactivity favours a model in which relaxed selective pressure in the ASC compartment is likely to be responsible for the emergence of autoreactivity observed in the ICU cohort.
This study established autoantibodies of substantial affinity can arise at the onset of the humoral immune response, and likely play a greater role in robust autoreactive phenotypes than their preformed counterparts in severe COVID-19 cases. While at a cellular level, the acute expansion of naive IgG1 ASCs appeared to peak during severe infection and subsequently subside, kinetic analysis of serology samples presented a more nuanced picture. Despite general declines, autoreactivity may persist at significant levels, and even increase post infection, in certain patients.
The detection and monitoring of these de novo autoantibodies present good avenues for disease stratification, prognostic determination, and treatment approaches in patients. Consequently, the origins of autoreactivity in COVID-19 has been an important area of research. This study explores the origins and breadth of de novo autoreactivity in severe COVID-19, with the successful application of SPR and other advanced techniques to demonstrate its potential for early intervention and treatment of patients with post-COVID chronic autoimmunity.
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