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Anti-Saccharomyces cerevisiae antibodies in patients with COVID-19

Background and study aim Anti-Saccharomyces cerevisiae antibodies (ASCA) have been described in many autoimmune diseases (AIDs). Coronavirus disease 2019 (COVID-19) could trigger AIDs. This study aimed to determine the frequency of ASCA in patients with COVID-19. Patients and methods This study included 88 adult patients with severe COVID-19, 51 mild COVID-19, and 160 healthy blood donors. ASCA of isotype immunoglobulin (Ig)G and IgA were detected by enzyme-linked immunosorbent assay. Results The frequency of ASCA (IgG or IgA) was significantly higher in patients with severe COVID-19 (21.6 % vs 3.7 %, p < 10−3) and in patients with mild COVID-19 than in the healthy controls (13.7 % vs 3.7 %, p = 0.03). ASCA-IgA was significantly more frequent in patients with severe COVID-19 than in healthy controls (15.9 % vs 0.6 %, p < 10−3). ASCA-IgG was significantly more frequent in patients with mild COVID-19 than in healthy controls (13.7 % vs 3.1 %, p = 0.02). ASCA (IgG or IgA) were more frequent in severe than in mild COVID-19, but the difference was not statistically significant (21.6 % vs 13.7 %). ASCA-IgA was significantly more frequent in patients with severe than those with mild COVID-19 (15.9 % vs 0 %, p = 0.003). The mean ASCA-IgG and ASCA-IgA levels were significantly higher in patients with severe COVID-19 than in healthy controls (5.8 U/mL ± 11.8 vs 2.3 U/mL ± 2.8, p < 10−3 and 9.2 U/mL ± 21.5 vs 3.4 U/mL ± 1.7, respectively, p < 10−3). The mean ASCA-IgG levels were significantly higher in patients with mild COVID-19 than in healthy controls (6.2 U/mL ± 12.9 vs 2.3 U/mL ± 2.8, p < 10−3). The mean ASCA-IgA levels were significantly higher in patients with severe than in those with mild COVID-19 (9.2 U/mL ± 21.5 vs 2.6 U/mL ± 1.2, p = 0.03). Conclusion ASCA was more frequent in patients with COVID-19 than in healthy controls.

Saccharomyces cerevisiae (S. cerevisiae) is a yeast that is used for bread baking and is one of the predominant yeast in our mycobiota [10]. anti-S. cerevisiae antibodies (ASCA) are directed against the phosphopeptidomannan, a part of the cell wall of S. cerevisiae. ASCA is considered one of the serological markers of Crohn's disease [11][12]. Combined with perinuclear anti-neutrophil cytoplasmic antibodies, ASCA has been reported as a valuable marker to discriminate between Crohn's disease and ulcerative colitis [13]. Additionally, ASCA has been detected in many AIDs [14][15][16][17][18][19]. However, to our knowledge, only one study has determined ASCA in COVID-19 [20]. Therefore, the present study aimed to determine the frequency of ASCA in this viral disease, which could induce AIDs.

Patients
We enrolled two groups of patients with a confirmed COVID-19 diagnosis by real-time polymerase chain reaction test (RT-PCR) of an oropharyngeal swab sample from December 2020 to February 2021. The first group consisted of 88 adult patients with severe COVID-19 and the second group was composed of 51 adults with mild COVID-19. Group classification was based on clinical symptoms, respiratory rate, and oxygen saturation. COVID-19 severity was categorized under the World Health Organization Clinical Progression Scale [21]. All sera samples in the first group were collected after hospital admission, whereas patients in the second group were not hospitalized and were given health care at home, and their sera samples were collected at least 14 days after RT-PCR. Medical criteria for hospital admission were severe dyspnea, oxygen saturation on room air of ≤ 94 % or increase respiratory rate (≥30 breaths per min), confusion or altered mental status or comorbid condition with worsening shortness of breath, fever, and/or increasing cough. Patients with Crohn's disease or autoimmune disease were excluded from our study.
Sera of 160 blood donors were included as normal controls. All sera were stored at − 80 • C until use. The study was approved by the local ethics committee and all patients gave their informed consent.

Laboratory measurements
ASCA immunoglobulin (Ig)G and IgA were detected by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Orgentec Mainz® Germany). The antigen consisted of highly purified mannan from S. cervisiae. Results were expressed as arbitrary units with a cut-off for positivity of 10 U/mL following the manufacturer's instructions.

Statistical analysis
The comparison of frequencies of ASCA was performed using Chisquare or Fisher's test. We used a parametric Student's t-test to compare the mean titer of ASCA. A Pearson correlation test was used to show the relationship between variables. A p-value of < 0.05 was considered significant. Table 1 shows the epidemiologic characteristics of patients and healthy controls.

Comparison of the frequency of ASCA between patients with severe and mild COVID-19
ASCA (IgG or IgA) were more frequent in severe than in mild COVID-19, but the difference was not statistically significant (21.6 % vs 13.7 %). ASCA-IgA was significantly more frequent in patients with severe than in mild COVID-19 (15.9 % vs 0 %, p = 0.003) ( Table 2).

Discussion
The present study demonstrated a significantly higher ASCA frequency in both patients with mild and severe COVID-19 than in healthy controls. These results confirm those of Sacchi et al. [20] who determined ASCA in a group of symptomatic and hospitalized patients with COVID-19. We found that IgA was the predominant isotype of ASCA in patients with severe COVID-19 (15.9 % vs 9 %). ASCA-IgA was more frequent than ASCA-IgG (25 % vs 17.5 %) even in the study of Sacchi et al. [20]. However, in all AIDs in which we previously detected ASCA [14][15][16][17][18][19] and in inflammatory bowel disease [22], IgG was the predominant isotype (Table 3). The discrepancy between AIDs and this severe viral infection could be because SARS-CoV-2-induced mucosal damage preferentially stimulates the IgA immune response [5]. Severe COVID-19 was associated with elevated serum IgA [23].
The frequency of ASCA-IgA in our patients with severe COVID-19 is lower than that of Sacchi et al. [20] (15.9 % vs 25 %). The difference  between these results could be explained by both the method used for ASCA assay and the number of patients studied. We used ELISA to determine ASCA and Sacchi et al. [20] used fluorescent enzyme immunoassay. Moreover, we included 88 patients in our study but Sacchi et al [20] had only 40 patients.
Only the IgG isotype of ASCA was detected in our patients with mild COVID-19 in contrast to those with severe COVID-19. This is because serum sampling has been done at the hospital admission for patients with severe COVID-19 and at least 14 days after RT-PCR analysis for those with mild COVID- 19. IgA has been demonstrated to dominate the early neutralizing antibody response to SARS-CoV-2 [24], while IgG could be detected by day 14 after the onset of symptoms [25]. Additionally, severely SARS-Cov-2-infected patients sustained increased IgA SARS-CoV-2 antibodies compared with those with mild COVID-19 who demonstrated an immunodominant IgG [26]. The present study determined ASCA but not anti-SARS-CoV-2 antibodies; however, interestingly, cross-reactive epitopes exist between S. cerevisiae and SARS-CoV-2. The Nucleic Acid Binding Domain of SARS-CoV non-structural protein 3 (nsp3) particularly shows close similarity to the RNA-binding site of the sterile alpha motif of the S. cerevisiae Vts1p protein [27].
The present study revealed that ASCA were slightly more frequent in patients with severe COVID-19 than in those with mild COVID-19 (21.6 % vs 13.7 %). We hypothesized that when ASCA are produced by the immune system, they could neutralize S. cerevisiae which is a beneficial yeast for our health [28,29]. Beta-glucan, derived from S. cerevisiae, primes the immune system to respond better to any viral infection [30]. Additionally, the use of oral beta-glucan was hypothesized to boost immune responses and abrogate symptoms in COVID-19 [31]. Betaglucan is a polysaccharide that is abundantly found in the cell wall of S. cerevisiae [31], and ASCA is directed against the peptidomannan of the cell wall of S. cerevisiae.
The presence of ASCA in patients with COVID-19 could be explained by three mechanisms. First, gut microbiota dysbiosis has been demonstrated in patients with COVID-19 [32][33][34]. The consequent barrier dysfunction [35] will allow S. cerevisiae to reach the intestinal mucosa. The immune mucosa is therefore stimulated and ASCA is synthesized. Second, during COVID-19, the lectin pathway of complement activation is triggered by the binding of mannose-binding lectin (MBL) with SARS-CoV spike protein [36]. Elevated MBL plasma levels have been reported in patients with COVID-19, and it is associated with thrombosis and coagulopathy in patients with severeCOVID-19 [37]. Furthermore, the transmembrane spike glycoprotein of SARS-CoV-2 is covered with Nlinked glycan having oligomannose and complex sugars [38]. Thus, could we explain the high frequency of ASCA in patients with COVID-19 by an eventual molecular mimicry between mannan of the yeast cell surface of S. cerevisiae and mannan of MBL or mannose of SARS-CoV-2? Fascinatingly, plasma MBL levels are also high in AIDs, in which we have previously detected ASCA, particularly T1D, celiac disease, primary biliary cholangitis, and Grave's disease [18]. Third, a common epitope between S. cerevisiae and Candida albicans has been demonstrated [39]. Furthermore, a high risk factor for candidemia was found in patients with severe-COVID-19 [40]. Therefore, a high frequency of ASCA in patients with severe COVID-19 could also be explained by the high incidence of candidemia among these patients.
ASCA could explain some extrapulmonary manifestations of COVID-19. S. cerevisiae has structural similarities with many human autoantigens [41]. Remarkably, SARS-CoV-2 has also cross-reactive epitopes with many human proteins [5,42]. Thus, both ASCA and anti-SARS-CoV-2 antibodies could bind to self-antigens and initiate a complement activation, which has a multiorgan impact on COVID-19 [36]. Crossreactive epitopes between SARS-CoV and S. cerevisiae have been reported [27]. Hence, both anti-SARS-CoV-2 antibodies and ASCA could bind to SARS-CoV-2 or S. cerevisiae which translocates from the gut to other organs via the vascular compartment because of a leaky intestinal wall [35] secondary to gut microbiota dysbiosis.
In conclusion, we have demonstrated a high frequency of ASCA in patients with COVID-19 and revealed that IgA was the predominant isotype of ASCA in patients with severe COVID-19 while only IgG isotype of ASCA detected in patients with mild COVID-19.

Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent
Informed consent was obtained from all individual participants included in the study.