key: cord-0255718-548jvahq authors: Liu, Y.; Ebinger, J. E.; Mostafa, R.; Budde, P.; Gajewski, J.; Walker, B.; Joung, S.; Wu, M.; Bräutigam, M.; Hesping, F.; Schäfer, E.; Schubert, A.-S.; Zucht, H.-D.; Braun, J.; Melmed, G. Y.; Sobhani, K.; Arditi, M.; Van Eyk, J. E.; Cheng, S.; Fert-Bober, J. title: Paradoxical Sex-Specific Patterns of Autoantibodies Response to SARS-CoV-2 Infection date: 2021-07-19 journal: nan DOI: 10.1101/2021.07.15.21260603 sha: 782adeebdf08bf5287c51bfafe5d486dc7e6653d doc_id: 255718 cord_uid: 548jvahq Background. Amidst the millions of individuals affected directly by the pandemic, pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating not only the acute response but also recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, we deliberately examined sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. Methods. We used a bead-based array containing over 90 autoantigens previously linked to a range of classic autoimmune diseases to assess autoantibody (AAB) titers in 177 participants. All participants had confirmed evidence of prior SARS-CoV-2 infection based on presence of positive anti-nucleocapsid IgG serology results (Abbott Diagnostics, Abbott Park, Illinois). We used multivariate analysis to determine whether sex-bias was associated with increased rates of AABs reactivity and symptom burden after SARS-CoV2 infection. Results. 82.4% of AABs reactivity was associated with being male compared to 17.6% with female. We found a diversity of AABs responses that exhibited sex-specific patterns of frequency distribution as well as associations with symptomatology and symptom burden. Conclusion. Our results reveal a remarkable sex-specific prevalence and selectivity of AAB responses to SARS-CoV-2. Further understanding of the nature of triggered and persistent AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19. Mechanisms underlying sex differences in both susceptibility and response to SARS-CoV-2 infection remain poorly understood. Biological sex differences have become manifest with respect to vulnerability to infection, adaptive immune responses, and the equilibrium of inflammation and tissue repair in the resolution of infection (1) . Recent evidence points to the possible contributions of triggering and persistence of autoimmune activation in SARS-CoV-2-infected COVID-19 patients (2, 3) . Intriguingly, despite classic autoimmune diseases being more prevalent in females, emerging studies have revealed a paradoxical male predominance of autoimmune activation in the setting of severe COVID-19 illness (4) . The extent to which such paradoxical sex differences in triggered autoimmunity may exist and persist across the broader clinical spectrum of SARS-CoV-2 infection is unclear. Recognizing that sex bias is potentially introduced when assessing autoimmune activation in the setting of more severe forms of COVID-19 illness, we deliberately aimed to interrogate sex-specific autoimmune activation after SARS-CoV-2 exposure in the absence of any extreme manifestations of clinical disease. Therefore, using an array to detect autoantibodies (AABs) to over 90 antigens previously linked to a range of classic autoimmune diseases, we sought to comprehensively examine the diversity of AAB responses in male and female health care workers (HCWs) who were exposed to SARS-CoV-2 and experienced even minimal or no symptoms. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; https://doi.org/10.1101/2021.07.15.21260603 doi: medRxiv preprint 6 also detected in HCs, with trend of AABs reactivity much more pronounce in male however, the AABs that showed reactivity in the HCs group were different compare to AABs reactive in SLE and HCWs individuals (Figure 2) . In male among HCs AAB reactivity to colony stimulating factor 2 (CSF2), glycoprotein secreted by macrophages, Endothelin converting enzyme 1 (ECE1) and Dihydrolipoamide Branched Chain Transacylase E2 (DBT) involved in protein break down were the most dominated AABs. In female, among HCs reactivity to Transforming growth factor beta (TGFb) and Thyroid Peroxidase (TPO, marker for Hashimoto's disease or Graves' disease) showed the highest titer. By contrast, among asymptomatic individuals, the breadth and magnitude of AAB reactivity was much more pronounced in females compared to males ( Figure 2B) . Notably, AABs to cytokine and chemokine antigens (IL6 and CSF2) involved in immune defense, together with lung specific proteins (gastrin release peptide (GRP) and serpin family B member 3 (SERPINB3), were predominantly elevated in asymptomatic females. By contrast, thyroid stimulating hormone receptor (TSHR) and lysine demethylase 6B (KDMA6B), which are known primary antigens in autoimmune diseases, were highly expressed in asymptomatic males. Among all participants who had at least mild symptoms, the range and degree of AAB reactivity was more pronounced in males compared to females (Figure 2C-D) . In participants with more than mild symptoms, 82.4% of AABs reactivity was associated with being male compared to 17.6% with female. The most abundant AABs in males including classical nuclear AABs such as small nuclear ribonucleoprotein polypeptide C (SNRPC) 7 In age-adjusted regression analyses we examined the sex-specific associations of distinct AABs levels with symptomatology (one symptom and marker per model), as shown in (Figure 3) . Notably, in males, a large number of AABs had increased levels in relation to at least a mild overall symptoms burden while a substantial proportion of these same AABs exhibited lower levels in relation to asymptomatic status. By contrast, a smaller number of AABs exhibited generally consistently increased levels in relation to any level of overall symptom burden in females, including asymptomatic status. In males, among the significantly associated AABs, small nuclear ribonucleoprotein polypeptides B (SNRPB), chromodomain helicase DNA binding protein 4 (CHD4) and chromogranin A (CHGA) were the most frequently associated with the distinct symptoms of 8 To further investigate the AAB sub-groups and select potential discriminatory symptoms, we applied hierarchical clustering analyses to identify similar magnitudes and directions of associations across AABs. The results of sex-specific two-dimensional clustering of symptoms variables in relation to AABs are shown in Figure 4 . In males, the initially identified cluster included the symptoms of muscle aches and fatigue (cluster 1), with diarrhea and loss of appetite clustered next (cluster 2), and sneezing, runny nose, and nasal congestion also clustered together (cluster 3). In females, there were also three major clusters identified: dry cough, chilis, and loss of appetite (cluster 1); sore throat, nausea, nasal congestion, and fever (cluster 2); and smell/taste change and shortness of breath (cluster 3). In males, clustering of AABs including C3 with TG antigens (cluster 1) and AABs to antigens representing LYZ and IFNA6 protein (cluster 2). In females, we found 5 clusters in total: ECE1 and HARS (cluster 1); SMD3, UBTF, and TOP1 (cluster 2); IL10 and S100A9 (cluster 3); HIST1H4A, MX1, and EXOSC10 (cluster 4); and, RPLP2 and TG (cluster 5). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In this study, comprehensive profiling of AAB activation in over 170 HCs with prior SARS-CoV-2 infection revealed several important sex-specific findings of interest. First, a surprisingly large number of the diverse autoantibodies assayed were differentially activated in males compared to females. Among previously infected individuals who were asymptomatic, the breadth of AABs response was more prominent in women than in men; by contrast, among previously infected individuals who experienced at least a mild burden of symptoms, the extent of antibody response was far more pronounced in men. Second, we found that the AABs response to symptom clusters were also sex-specific, with certain AABs-symptom associations seen more prominently in men compared to women, across the range of symptom burden. Finally, we observed these sex-specific AABs associations up to 6 months following symptomatology, indicating that SARS-CoV-2 triggers a complement of AABs responses that persists over time -in a sex-specific manner and irrespective of illness severity. The current study expands from prior work in several ways. Extending from previously studies reporting on presence of post-COVID-19 autoimmunity, (3, 7) we employed a broad array of antibodies to over 90 distinct antigens previously linked to classic autoimmune conditions. Our results reveal a remarkable sex-specific prevalence and selectivity of the AABs response to SARS-CoV-2. Confirming and extending from the findings from prior reports, we found that a majority of our previously infected study participants had detectable circulating AABs against antigens such as ACE2, AQP4, C3, CHD4, CHGA, CXCL8, DBT, ECE1, ELANE, EXOSC10, HARS, HIST1H4A, IGF1R, INS, MOV10, MX1, PRTN3, RNF41, RPLP2, S100A9, SET, SNRPD1, SOX13, TG, TGFB1, TOP1, UBTF. Intriguingly, a distinct set of AABs to 59 antigens were highly correlated with reported symptoms in the male population, while another set of AABs to only 38 antigens were associated with symptoms in females. Notably, in males, we observed AABs associated with symptoms at a high All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; https://doi.org/10.1101/2021.07.15.21260603 doi: medRxiv preprint frequency (≥6 symptoms) as well as at a moderate frequency (≥4 symptoms). The high frequency associated AABs included SNRPB, a ribonucleoprotein that is widely prevalent in human SLE (8) . The moderate frequency associated AABs included MOV10, CHGA, CHD4, HIST1H4A, ACE2, IFNA6, LYZ, RNF41. Importantly, both MOV10 and IFNA6 have been reported in patients infected with COVID-19 (5, 9) . In females, we observed an overall lower frequency of significant symptoms associated AABs when compared to males. The 3 most prominent symptoms in females were associated with AABs to DBT and ROS1. Interestingly, AABs to DBT have been associated with lung cancer (10). Importantly, a number of AABs can be classified as implicated more frequently with systemic disease traits (i.e., multi-organ or multi-system) which may be particularly relevant to the more non-specific symptoms such as fatigue, fever, rashes, cold or allergy-type symptoms, weight loss, and muscular weakness. The sex specificity of triggered AABs reactivity in association with either distinct symptoms, or symptom clusters, may be related not only to sex differences in acute illness but also in post-acute and chronic clinical syndromes experienced by a substantial number of individuals recovering from COVID-19 (11). While apparently paradoxical at the outset, our sex-specific findings are congruent with ongoing emerging data regarding potential mechanisms underlying sex differences in the susceptibility and response to SARS-CoV-2. Early studies reported that while men and women have similar prevalence, men with COVID-19 are at greater risk for worse outcomes and death independent of age (12, 13). Consistent with these findings, conventional inflammatory markers are founded to be more substantially elevated in men compared to women who are hospitalized for COVID-19 (14) . Accordingly, males in our study had a greater than 1.5-fold odds of AABs reactivity after adjusting for age. For classic autoimmune disease, clinical prevalence and incidence of autoimmune diseases tends to follow a male versus female pattern based on pathology. Male-predominant autoimmune diseases usually manifest clinically (i.e., show signs and symptoms of clinical disease) prior to age 50 and are characterized by acute inflammation and a Th1-type response, whereas autoimmune All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. diseases with a greater incidence in females that occur early in life have a clearer antibodymediated pathology. Autoimmune diseases that have a greater incidence in females and also appear clinically later in life tend to present with evidence of chronic pathology, fibrosis, and increased numbers of autoantibodies are present (15) . The conventional sex bias seen for classic autoimmune diseases has been attributed in part to women who have a generally stronger cellular and humoral immunity, higher levels of circulating antibodies, more numerous circulating CD4+ T cells, and more robust cytokine production in response to immune stressors such as infection (16, 17) . By contrast, males are now recognized as more vulnerable to the immune-modulated effects of active SARS-CoV-2 infection likely due to multiple mechanisms (e.g. lower immune cell expression of TLR7, lower observed antibody response, and lower interleukin mediated tissue resilience and tissue repair activity)(17) -and our results demonstrate the persistence of detectable downstream autoimmune sequelae. Importantly, our study also demonstrates for the first time a persistence of autoimmune activation in females compared to males following asymptomatic infection. As context, the stability of AABs in classic autoimmune diseases is known to vary substantially, with some autoantibodies fluctuating with flares of disease, while others remain stable. We can speculate that the preponderance of AABs positivity in females -in the absence of symptomatic or recognized infection -represents initiation or proliferation and then persistence of self-reactive immunity with implications for post-acute chronic immune-driven disease states. These findings may be particularly relevant to rapidly accumulating evidence of the post-acute SARS-CoV-2 syndromes (e.g. "long-haul COVID") that can emerge even weeks to months following resolution of mild or asymptomatic infection and with clinical manifestations that appear to differ in women compared to men (11). The existence of autoantibodies within normal healthy individuals has been already shown by other investigators (18). The fact that across the breadth of AABs assayed in our healthy control sample, titers were also male predominant suggesting that larger population-based screening studies are needed to clarify our understanding of sex differences in basal AAB variation in the absence of clinical disease. Importantly, variations in the AAB titers found in the HCWs were different than those seen in healthy control subjects. In the latter group, the most dominant AAB was for granulocyte-macrophage colony-stimulating factor, also known as colony-stimulating factor 2 (CSF2), well-known to be a regulator of monocyte/macrophage differentiation. By contrast, AAB against CSF2 in the HCWs was barely reactive in the male population and were seen to be upregulated in female in asymptomatic group. Several limitations of our study merit consideration. Our cohort includes HCWs from a single center who volunteered and responded to surveys, potentially limiting generalizability. We have a relatively small number of male subjects (n=63) that may have limited the ability to detect potential additional predicators of post-COVID autoimmunity; thus, further investigations of larger sized samples are needed. Although this was a prospective study, the survey method involved requesting participants to self-report symptoms occurring up to 6 months prior to the blood draw, contributing to potential recall bias. Whether examined subjectively or objectively, symptomatology can vary not only between but also within individuals over time. Similarly, the status of AAB reactivity may change over time and in relation to the timing of initial or repeated exposures. Thus, future longitudinal studies are warranted to understand temporal trends in similarly measured exposure and outcomes. In summary, this comprehensive study of AABs to a wide array of antigens found that male sex carries the risk of diverse autoimmune activation following symptomatic COVID-19 illness, whereas female sex carries risk for a distinct profile of autoimmune activation following asymptomatic SARS-CoV-2 exposure. Importantly, both sets of sex-specific AAB All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; 13 reactivity patterns were found to persist up to 6 months following associated symptomatology. Further understanding the nature of triggered and persistent AAB activation among individuals who are exposed to SARS-CoV-2 -and vulnerable to its potentially morbid clinical sequelae -will be essential for developing effective interventions and therapeutics. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. (Table S1 ). Coupling efficiency was confirmed by incubation of 625 beads from each coupled region with a phycoerythrin-conjugated anti-6× HisTag antibody (Abcam, Cambridge, UK) at a concentration of 10 μ g/mL for 45 min shaking at 900 rpm and room temperature. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; Coupled beads were mixed to a final concentration of 62.5 beads/μL and stored in PBS supplemented with 1% bovine serum albumin, 0.1 % Tween 20 and 0.05% ProClin™ 300 (Merck KGaA, Darmstadt, Germany), at 4 °C until use (8) . For analysis, serum or plasma samples were diluted 1:100 in assay buffer (50 % PBS with 1 % BSA, 50% LowCross-Buffer® (Candor Biosciences, Wangen, Germany), 1.3 µg/µl E. coli lysate) and incubated for 20 min at room temperature. Next, the bead's mix (50 Statistics. Parametric tests and non-parametric tests were used to compare normally distributed continuous variables and non-normally distributed or categorical variables, respectively. Histograms were used to display distribution of symptomatology as well as AABs reactivity against each antigen for the cohort in sex-pooled and sex-specific analyses. Ordinal logistic regression was used to examine the associations between AABs reactivity of each antigen and self-reported symptoms burden, defined as a symptom's severity score. The symptoms burden score was constructed based on the total number of reported All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; symptoms experienced within 6 months prior to the blood draw wherein a greater number of symptoms corresponded to a higher score (i.e., one point per symptom): we defined asymptomatic as represented by a score of 0, mild symptom burden as a score of 1 to 7, and more than mild symptom burden as a score of >7. All statistical analyses were conducted using R (v3.5.1) and statistical significance was defined as a two-tailed P value <0.05. Study approval. All participants provided written informed consent and all protocols were approved by the Cedars-Sinai institutional review board. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. We are grateful to all the front-line HCWsin our healthcare system who continue to be dedicated to delivering the highest quality care for all patients. We would like to thank the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In our primary study cohort, the distribution of experienced symptoms was generally similar between men and women (Panel A) with some exceptions including certain distinct symptoms being more frequent in men (e.g., chills, fever, conjunctivitis) and other distinct symptoms being more frequent in women (e.g., loss of appetite, nausea). In analyses of overall symptom burden, frequencies of asymptomatic, mildly symptomatic, and more than mildly symptomatic persons were relatively equally distributed between the sexes (Panel B). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; 22 autoantibodies and h=1.5 for symptoms from Ward hierarchical clustering. Results are shown for men in Panel A and women in Panel B. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table S6 . Associations of AABs reactivity with symptoms in women. P values from age-adjusted regression analysis comparing males with a specific symptom burden to females without the same symptom are shown. Last three columns show p values from age-adjusted regression analysis comparing females with different levels of symptoms burdens to the pre-pandemic healthy control group. Figure S1 . Sex-specific associations of autoantibodies with SLE status. In ageadjusted regression analyses, the breadth and magnitude of associations observed AABs reactivity and systemic lupus erythematosus (SLE) compared to health control status were predominantly seen in women compared to men. Beta coefficients were shown to the left, and negative log P values were shown to the right in each panel. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 19, 2021. ; https://doi.org/10.1101/2021.07.15.21260603 doi: medRxiv preprint Considering how biological sex impacts immune responses and COVID-19 outcomes Prevalence of autoantibody responses in acute coronavirus disease 2019 (COVID-19) Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls Molecular and Biological Mechanisms Underlying Gender Differences in COVID-19 An Autoantigen Profile of Human A549 Lung Cells Reveals Viral and Host Etiologic Molecular Attributes of Autoimmunity in COVID-19. bioRxiv : the preprint server for biology Introducing Polyautoimmunity: Secondary Autoimmune Diseases No Longer Exist Diverse Functional Autoantibodies in Patients with COVID-19 Key autoantigens in SLE Sex differences in inflammatory markers in patients hospitalized with COVID-19 infection: Insights from the MGH COVID-19 patient registry Sex differences in autoimmune disease from a pathological perspective Sex bias in autoimmunity. Current opinion in rheumatology Implications of Sex Differences in Immunity for SARS