Autoimmune anti-DNA and anti-phosphatidylserine antibodies predict development of severe COVID-19

COVID-19 induces high levels of autoimmune anti-DNA and anti-phosphatidylserine antibodies that are detected in some patients upon hospital admission and predict later development of severe disease.

1) While mentioned serval times throughout the text, it would be extremely informative to show, side-by-side, the levels of anti-RBCL, anti-PS, and anti-DNA found in COVID-19 patients alongside other infections and autoimmune diseases (such as SLE and APS) as well as provide a better description of their antigen specificity. In essence, what is the difference, if any, between anti-RBCL, anti-PS, and anti-DNA autoantibodies found in COVID-19 patients and other disease states? Information regarding autoantibody subtypes, in addition to IgG, is also desirable.
2) The circulating levels of IC reported in Figure 2 are surprisingly high for the control group. Also, their relationship with autoimmunity is unclear. Please explain.
3) COVID-19 patients were stratified into three groups: non-severe, severe patients who survived, and severe patients who died of COVID-19. However, no information is provided regarding their clinical history, specifically co-morbidities and medications, which is highly relevant for the search for autoantibodies. 4) For the anti-PS ELISA assay, after immobilization, PS-coated plates were washed 3 times with PBS 0.05% Tween-20 before blocking. Tween-20 is a nonionic detergent that is typically used for lysing cells as it destabilizes membrane bilayers. The use of Tween-20 is not reccomended when investigating protein-lipid interactions. Please explain why the authors use Tween-20 in their ELISA assays and, if possible, provide evidence that Tween-20 does not interfere with the detection of anti-PS autoantibodies.

Referee #2 Review
Comments on Novelty/Model System for Author: The study in my opinion is of clinical relevance. It describes for the first time that anti-DNA autoantibodies may play an important role in the pathogenesis of COVID-19 and could be developed as a predictive biomarker for disease severity and specific clinical manifestations. Anti-DNA antibodies correlate with a marker of thrombosis, Ddimer. Anti-DNA antibodies also correlated with a parameter that determines the variation of erythrocyte volume, RDW, which has been associated with mortality risk in multiple diseases, including COVID-19. These findings suggest that the most severe forms of disease observed in COVID-19 patients may be a result of the host response to infection, rather than a direct consequence of viral cytopathic effect. Autoantibodies, as part of the host response to infection, may contribute to this delayed pathogenesis through different mechanisms. Our work suggests that anti-DNA antibodies may have a role in different pathogenic processes, including cell injury and coagulation, constituting a possible mechanism contributing to pathogenesis in COVID-19 patients.
Remarks for Author: In this study the Authors performed a retrospective study of 115 COVID-19 hospitalized patients with different severity to analyze the generation of autoantibodies to common antigens: a lysate of erythrocytes, the lipid phosphatidylserine (PS) and DNA. The statistical relation of autoantibody levels to death, disease severity and specific pathologies was determined using high-throughput data analysis of 118 clinical parameters for all patients. They found that high levels of IgG autoantibodies against erythrocyte lysates were observed in a large percentage (up to 41%) of patients with COVID-19 compared to uninfected controls. Interestingly, they found that anti-DNA antibodies determined upon hospital admission were high in 16% of patients and correlated strongly with later development of severe disease, showing a positive predictive value of 89.5% and accounting for 22% of total severe cases. A statistically signifcant correlation was found between anti-DNA antibodies and markers of cell injury, coagulation, neutrophil levels and erythrocyte size. They concluded that Anti-DNA autoantibodies may play an important role in the pathogenesis of COVID-19 and could be developed as a predictive biomarker for disease severity and specific clinical manifestations.
The study is of interest and of clinical relevance. However, some issues deserve further details. Patients characteristics: how the disease severity was defined? which criteria? chest CT? clinical? Autoimmune antibodies: please specify if also other autoantibodies were searched for. To further improve the manuscript, in introduction or discussion, it would be interesting to discuss the results of the only study presently reporting the presence of autoantibodies in patients with COVID-19 possibly reflecting a pathogenetic role of immune dysregulation (COVID-19 and immunological dysregulation: can autoantibodies be useful? Clin Transl Sci. 2020 Sep 29:10.1111/cts.12908. doi: 10.1111/cts.12908). A relevant issue is the detection of anti-DNA antibodies since it is well known that solid-phase assays are less specific than indirect immunofluorescence technique using Crithidia luciliae (Diagnostic role of anti-dsDNA antibodies: do not forget autoimmune hepatitis. Nat Rev Rheumatol. 2021 Jan 18. doi: 10.1038/s41584-021-00573-7. Epub ahead of print). In the discussion, I suggest adding a paragraph discussing the potential limit of the used assay.

Referee #3 Review
Comments on Novelty/Model System for Author: There are concerns regarding the anti-PS & anti-DNA assays (see details in the report to the authors).
Remarks for Author: TThe paper reports a retrospective study on the presence of autoantibodies and circulating immune-complexes (CIC) in 115 COVID-19 hospitalized patients. The authors detected autoAbs against RBC-lysates, anti-PS & anti-DNA IgG and CIC by home-made assays. High levels of IgG autoAbs against RBC lysates were found in up to 41% of COVID-19 patients compared to uninfected controls. Anti-DNA Abs were detected in 16% of patients and correlated with later development of severe disease, markers of cell injury, coagulation, neutrophil levels & RBC size. The main authors' conclusion is that autoimmune responses may play a role in the pathogenesis of COVID-19 and that anti-DNA autoAbs may be a biomarker for disease severity.
Main comments 1. As stated by the authors themselves, different infections (viral, bacterial & protozoan) may induce autoaAbs against self-Ag. To support the authors' conclusions a control group of patients with another non-SARS-CoV-2 infection should be tested for the same autoAbs. 2. It seems that 20 uninfected normal (?) control samples were used for calculating the cutoff values of the home-made assays. A larger number is necessary and more information on age/sex etc distribution, how the normal values were calculated should be reported. For example, antiphospholipid antibodies do not display Gaussian distribution and cutoff values calculated as mean+ 3SD are not acceptable. 3. The method for the detection of anti-PS Abs is not in line with the usual methods for the antiphospholipid (aPL) antibodies. The described solid-phase detects Abs against PS and not against the PL-binding proteins (i.e. beta2GPI or prothrombin) which are actually the true autoimmune aPL. In this regard, any speculation on the significance of these autoAbs cannot be related to the antiphospholipid syndrome or even to the thrombophilic state supported by autoimmune aPL. Further experiments to investigate the presence of true aPL (e.g. aCL, anti-beta2GPI or anti-PS/PT IgG/IgM) should be carried out. 4. Anti-DNA Abs: the described assay likely detects anti-single stranded DNA rather than antidouble stranded DNA. Control experiments with sera positive for anti-dsDNA should be carried out to characterize the specificity of the assay. This point is closely related to comment in point n.1. In fact, anti-ssDNA Abs are well known to be detectable in several infectious disease at variance of anti-dsDNA Abs found in SLE or SLE-like disease only.
Minor comment 1. If is true that circulating autoAbs are playing a role in COVID-19 pathogenesis, do authors have data showing complement activation in their patients? 2. More details how the severity of the COVID-19 disease was defined should be reported.
2nd Review Round

Referee #1 Review
Remarks for Author: The revised version of the manuscript has much improved. It now provides new important information enabling a more direct comparison between COVID-19 and other diseases, namely SLE and malaria, characterized by elevated titers of autoantibodies. While I applaud the authors for the effort and amount of work, because of this new data, it appears that the presence and titers of anti-RBCL, anti-PS, and anti-DNA antibodies are not a unique signature of COVID-19 patients; rather, they may represent a more general biomarker of autoreactivity. Furthermore, even though anti-DNA and anti-PS antibodies are (slightly) more elevated in severe COVID-19 patients than non-severe patients and controls, the differences between these groups are not striking. This observation is even more puzzling to interpret considering that, as written by the authors, the analyses were not adjusted for any other comorbidities or clinical history that might have impacted patient outcomes.
I have two additional comments: 1) The authors provide no direct evidence that these autoantibodies may play important roles in the disease pathogenesis, unlike in currently published work (DOI: 10.1111/jt h.15455) 2) In this context, the choice of malaria is unclear. COVID-19 is an RNA virus. One would have expected to see comparative data between COVID-19 and a similar RNA virus, not a parasite. Thank you for submitting your manuscript entitled "Autoantibodies to DNA and to phosphatidylserine correlate with development of severe COVID-19" to Life Science Alliance. As indicated in the decision letter from our partner journal, I invite you to submit a revised manuscript addressing the following Reviewer comments.
-Address Reviewer 1's comment suggesting that this signature may be a more general biomarker of autoreactivity, as well as Additional Comment #2, via Discussion. Comment #1 should be addressed via Discussion, but no additional experimental data is required.
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Response to reviewers
Address Reviewer 1's comment suggesting that this signature may be a general biomarker of autoreactivity, as well as Additional Comment #2, via Discussion. Comment #1 should be addressed via Discussion, but no additional experimental data is required.
Reviewer 1 comment on signature being a general biomarker of autoreactivity: We have observed that the levels of anti-RBC antibodies, which are actually a general biomarker for autoreactivity, do not correlate with or predict disease severity (Table III), indicating that the signature is not a general biomarker for autoreactivity. There is also evidence from other published work that while particular antibody specificities correlate with disease severity, levels of general autoreactivity do not (Wang et al., 2021). This is now mentioned in Discussion line 276.
Reviewer 1 comment on choice of malaria as comparison for autoreactivity: The original request of the reviewer was to compare the autoreactivity levels of COVID-19 to other infectious disease and did not specify RNA virus. The choice of malaria was made on: 1) the previous knowledge that, similar to COVID-19, malaria is highly inflammatory and triggers a strong autoantibody response and 2) sample availability. The similarities between malaria and COVID-19 are highlighted on Discussion line 255.
We have modified the title to indicate more precisely that anti-DNA and anti-PS predict disease severity, since antibody determinations were made at the time of admittance to the hospital but severity was developed later in patients. Thank you for submitting your revised manuscript entitled "Autoimmune anti-DNA and anti-Phosphatidylserine antibodies predict development of severe COVID-19". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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