key: cord-0858580-12prczym authors: Urra, José Miguel; Ferreras-Colino, Elisa; Contreras, Marinela; Cabrera, Carmen M.; Fernández de Mera, Isabel G.; Villar, Margarita; Cabezas-Cruz, Alejandro; Gortázar, Christian; de la Fuente, José title: The antibody response to the glycan α-Gal correlates with COVID-19 disease symptoms date: 2020-07-14 journal: bioRxiv DOI: 10.1101/2020.07.14.201954 sha: ce38519716294953b5253afa465c6a9cecaab701 doc_id: 858580 cord_uid: 12prczym The coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. The characterization of the immunological mechanisms involved in disease symptomatology and protective response is important to advance in disease control and prevention. Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the development of a protective response against pathogenic viruses and other microorganisms containing this modification on membrane proteins mediated by anti-α-Gal IgM/IgG antibodies produced in response to bacterial microbiota. In addition to anti-α-Gal antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms that have shown protection in animal models against infectious diseases without inflammatory responses. In this study, we hypothesized that the immune response to α-Gal may contribute to the control of COVID-19. To address this hypothesis, we characterized the antibody response to α-Gal in patients at different stages of COVID-19 and in comparison with healthy control individuals. The results showed that while the inflammatory response and the anti-SARS-CoV-2 (Spike) IgG antibody titers increased, reduction in anti-α-Gal IgE, IgM and IgG antibody titers and alteration of anti-α-Gal antibody isotype composition correlated with COVID-19 severity. The results suggested that the inhibition of the α-Gal-induced immune response may translate into more aggressive viremia and severe disease inflammatory symptoms. These results support the proposal of developing interventions such as probiotics based on commensal bacteria with α-Gal epitopes to modify the microbiota and increase the α-Gal-induced protective immune response and reduce the severity of COVID-19. The coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory 25 syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. The 26 characterization of the immunological mechanisms involved in disease symptomatology and 27 protective response is important to advance in disease control and prevention. Humans evolved 28 by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which 29 resulted in the development of a protective response against pathogenic viruses and other 30 microorganisms containing this modification on membrane proteins mediated by anti-α-Gal 31 IgM/IgG antibodies produced in response to bacterial microbiota. In addition to anti-α-Gal 32 antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms 33 that have shown protection in animal models against infectious diseases without inflammatory 34 responses. In this study, we hypothesized that the immune response to α-Gal may contribute to 35 the control of COVID-19. To address this hypothesis, we characterized the antibody response 36 to α-Gal in patients at different stages of COVID-19 and in comparison with healthy control 37 individuals. The results showed that while the inflammatory response and the anti-SARS-CoV-38 2 (Spike) IgG antibody titers increased, reduction in anti-α-Gal IgE, IgM and IgG antibody 39 titers and alteration of anti-α-Gal antibody isotype composition correlated with COVID-19 40 severity. The results suggested that the inhibition of the α-Gal-induced immune response may 41 translate into more aggressive viremia and severe disease inflammatory symptoms. These 42 results support the proposal of developing interventions such as probiotics based on commensal 43 The coronavirus disease 19 (COVID-19), a pandemic caused by severe acute respiratory 47 syndrome coronavirus 2 (SARS-CoV-2), has rapidly evolved from an epidemic outbreak to a 48 disease affecting the global population. SARS-CoV-2 infects human host cells by binding to 49 the angiotensin-converting enzyme 2 (ACE2) receptor [1] . It acute respiratory failure who needed mechanical ventilation support were admitted to a hospital 110 ICU. The patients were discharged from the hospital due to the clinical and radiological 111 improvement of pneumonia caused by the SARS-CoV-2, along with the normalization of 112 analytical parameters indicative of inflammation, such as C-reactive protein (CRP), D-Dimer 113 and blood cell count (Table 1) . Samples from asymptomatic COVID-19 cases with positive 114 anti-SARS-CoV-2 IgG antibody titers but negative by RT-PCR (n = 10) were collected in May 115 22-29, 2020 and included in the analysis. Samples from healthy control individuals (n = 37) 116 were collected prior to COVID-19 pandemic in April 2019. The use of samples and 117 individual's data was approved by the Ethical and Scientific Committee (University Hospital 118 of Ciudad Real, C-352 and SESCAM C-73). 119 120 3.1. Inflammatory biomarkers are associated with severity in COVID-19 patients 192 193 In the blood cell analysis, the ICU patients showed higher lymphocytopenia, percentage and 194 neutrophil counts when compared to hospital discharge and hospitalized individuals (p < 0.001; 195 Figure 1a and Table 1 ). The cellular and biochemical indicators of systemic inflammation, 196 Neutrophil-Lymphocyte Count Ratio (NLR), C-reactive protein (CRP) and D-dimer levels 197 were higher in ICU patients when compared to other patients (p < 0.002; Figure 1a and Table 198 1). Although more severe symptoms have been associated with elderly patients, herein older 199 patients were recorded in the hospitalized and not the ICU group (p < 0.001; Table 1 The serum IgA, IgE, IgM and IgG antibody response to α-Gal was characterized in healthy 226 individuals and COVID-19 patients at different disease stages (Figures 2 and 3a) . A negative 227 correlation was observed for IgE, IgM and IgG between anti-α-Gal antibody titers and disease 228 severity (rs < 0; p = 0; Figure 3a) . The anti-α-Gal IgA antibody titers did not vary between the 229 different groups (p = 0.21136; Fig. 3a ) nor correlate with disease severity (rs = 0.02; p = 0.91; 230 Figure 3a ). For anti-α-Gal IgM and IgG antibodies, the titers decreased from healthy to ICU 231 individuals (p < 0.00001; Figures 2 and 3a) . However, in asymptomatic cases the anti-α-Gal 232 IgE titers were higher than in healthy individuals and symptomatic COVID-19 patients (p < 233 0.000001; Figure 3a ). In COVID-19 patients, the IgE but not IgM and IgG antibody titers were 234 higher in hospitalized patients than in hospital discharge and ICU cases (p < 0.05; Figure 2 ). The profile of anti-α-Gal antibody isotypes was qualitatively compared between groups 243 including reference values for serum immunoglobulin levels (Figure 3b ). The results evidenced 244 Gal antibody isotypes in COVID-19 cases that may be associated with different disease stages 294 ( Figure 5 ). These results suggested that higher anti-α-Gal IgE levels in asymptomatic cases 295 may reflect an allergic response mediated by this glycan, which reflects the trade-off associated 296 with the immune response to α-Gal that benefit humans by providing immunity to pathogen 297 infection while increasing the risk of developing allergic reactions to this molecule [12, 13, 17] . 298 In healthy individuals as in hospital discharge cases, the higher representation of anti-α-Gal 299 IgM and/or IgG antibodies may be associated with a protective response to COVID-19. 300 However, in hospitalized patients the representation of anti-α-Gal antibody isotypes did not 301 vary, which could reflect the absence of protection. Finally, the higher representation of anti-302 α-Gal IgA antibodies in ICU patients may be associated with the inflammatory response 303 observed in these cases. In accordance with these results, it was recently shown in endogenous 304 α-Gal-negative turkeys that treatment with probiotic bacteria with high α-Gal content results 305 in protection against aspergillosis through reduction by still unknown mechanisms in the pro- Based on the fact that natural antibodies against α-Gal are produced in response to bacteria 317 with this modification in the microbiota [10], our hypothesis is that the dysbacteriosis observed 318 in COVID-19 patients [28] translates into a reduction in total anti-α-Gal antibody titers and 319 alteration of anti-α-Gal antibody isotype composition due to the reduction in the microbiota of 320 α-Gal-containing commensal bacteria and other still uncharacterized mechanisms ( Figure 5) that may be implicated in the human protection to COVID-19. 342 343 In conclusion, according to these results and previous findings in retrovirus [41, 42] , The authors declare that there is no conflict of interest regarding the publication of this paper. 355 (University of Castilla La Mancha, UCLM, Spain) for the critical reading of the manuscript. 363 We acknowledge UCLM, Spain support to Grupo SaBio. MC was funded by the Ministerio de 364 Ciencia, Innovación y Universidades, Spain (grant FJC-2018-038277-I). IGFM was supported 365 by the UCLM. 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