Review 1: "Broadly-targeted autoreactivity is common in severe SARS-CoV-2 Infection"

John P. Hussman

doi:doi:10.1162/2e3983f5.afd87055

Relaxed peripheral tolerance drives broad <i>de novo</i> autoreactivity in severe COVID-19

by Matthew C. Woodruff, Richard P. Ramonell, Ankur Singh Saini, Natalie S. Haddad, Fabliha A. Anam, Mark E. Rudolph, Regina Bugrovsky, Jennifer Hom, Kevin S. Cashman, Doan C. Nguyen, Shuya Kyu, Michael Piazza, Christopher M. Tipton, Scott A. Jenks, F. Eun-Hyung Lee, and Ignacio Sanz

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AbstractAn emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of de novo autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone IGHV4-34 expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.

RR:C19 Evidence Scale rating by reviewer:

Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

The brief report by Woodruff et al. identifies the generation of autoreactive antibodies in patients with COVID-19. This is a potentially important aspect of SARS-CoV-2 infection, as autoreactive antibodies directed toward targets including IFN-I, phospholipids, and recently ACE2, have been reported by several groups in association with severe COVID-19. The paper might usefully include references to Zhang et al.¹ and Casciola-Rosen et al.² in this regard.

The key finding reported here is the presence of anti-nuclear antibody (ANA) at elevated titers (1:160 and above) in the majority of patients with severe/critical COVID-19, as well as autoantibodies against rheumatoid factor (RF), phospholipids, and other self-antigens. Moreover, ANA and anti-RF production is reported to be correlated with C-reactive protein (CRP) level.

The authors are encouraged to note in the text that low titer ANAs are commonly observed in healthy controls and may be benign, without implying autoimmune pathology. Thus the autoantibody high titer and correlation with CRP reported here are meaningful. The finding that over half of patients displaying ANA also generated autoreactive antibodies to at least one other antigen also helps to distinguish the findings reported here from the presence that might otherwise be benign. Greater emphasis on these points may help to avoid misplaced concern about the mere presence of ANAs in patients with COVID-19.

As elevated ANA is more common in females than males, and is correlated with vitamin D levels and various other factors³, the presence of autoreactive antibodies across such classifications would also be interesting, but not essential, to report.

The brief discussion includes a proposal that increased activation of TLR7 by ssRNA may contribute to the observed break in immune tolerance. As TLR hypersensitivity is also observed in EBV, resulting in the activation of autoreactive B-cells⁴, this mechanism appears to be a reasonable possibility. The potential contribution of additional mechanisms such as molecular mimicry might also be mentioned.

The paper is well written, with only minor edits required, and the graphical elements are appropriate. Abbreviations for terms such as an antinuclear antibody, ribonucleoprotein, and C-reactive protein, among others, should follow the explicit introduction of these terms. The two appearances of the word “it’s” in the final sentence need no apostrophe.

With respect to areas that might benefit from additional analysis, the central question is the extent to which the presence of autoantibodies may have consequences for enhanced pathology. While not obligatory, it may be informative to examine and report further associations between autoantibody titer and clinical markers such as IL-6, complement activation, and lactate dehydrogenase (LDH), as well as the relative frequency of coagulopathy and thrombosis between subgroups classified based on the presence of autoantibodies.

Given the brevity of the report, it is not possible to assess the methodological aspects of the research. The work appears reliable based on the strength of the evidence provided.

John P. Hussman, Ph.D., Director, Hussman Foundation, Ellicott City MD

1. Zhang Y, Cao W, Jiang W, et al. Profile of natural anticoagulant, coagulant factor and anti-phospholipid antibody in critically ill COVID-19 patients. J Thromb Thrombolysis 2020; 50: 580-586.

2. Casciola-Rosen L, Thiemann DR, Andrade F, et al. IgM autoantibodies recognizing ACE2 are associated with severe COVID-19. medRxiv 2020: 2020.2010.2013.20211664.

3. Grygiel-Górniak B, Rogacka N, Puszczewicz M. Antinuclear antibodies in healthy people and non-rheumatic diseases - diagnostic and clinical implications. Reumatologia 2018; 56: 243-248.

4. Wang H, Nicholas MW, Conway KL, et al. EBV latent membrane protein 2A induces autoreactive B cell activation and TLR hypersensitivity. J Immunol 2006; 177: 2793-2802.