key: cord-0716956-2miafaki
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: 2020-10-30
journal: J Med Virol
DOI: 10.1002/jmv.26575
sha: 2ccc56450ba2e0249cdec605fe40b7aeae2d8a7c
doc_id: 716956
cord_uid: 2miafaki

The coronavirus disease 2019 (COVID‐19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has affected millions of people worldwide. Characterization of the immunological mechanisms involved in disease symptomatology and protective response is important to progress 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 immunoglobulin M (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 α‐Gal‐induced protective immune response and reduce severity of COVID‐19.

The coronavirus disease , a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly evolved from an epidemic outbreak to a disease affecting the global population. SARS-CoV-2 infects human host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. 1 It has been established that COVID-19 mainly affects the respiratory tract, but as a systemic disease, it affects multiple processes including the gastrointestinal, cardiovascular, neurological, hematopoietic, and immune systems. 2 Several days after the onset of symptoms, the SARS-CoV-2 infection becomes more systemic and affects various organs with inflammatory responses and lymphocytopenia. 2 Lymphocytopenia is likely caused by the direct lethal effect of SARS-CoV-2 on lymphocytes with the ACE2 receptor 3 and the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1) and IL-6 that induce apoptosis in lymphocytes. 4 The "cytokine storm syndrome (CSS)" has been associated with COVID-19 through the activation of the nuclear factor-kB (NF-kB) innate immune pathway resulting in the upregulation of proinflammatory cytokines. 5 Lymphocytopenia in patients with along with the rise in neutrophils has been associated with worse disease prognosis. Consequently, patients with respiratory distress syndrome in intensive care unit (ICU) show lower lymphocyte counts and higher mortality when compared to other COVID-19 patients. 6, 7 Additionally, COVID-19 patients suffer dysbacteriosis in the gut and lung microbiota due to enrichment of opportunistic pathogens and depletion of beneficial commensals, which recommends the development of interventions such as probiotics to reduce the severity of COVID-19 through modification of the microbiota composition. 1, 8, 9 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 of anti-α-Gal IgM/IgG antibodies against pathogenic viruses (e.g., HIV), bacteria (e.g., Mycobacterium) and parasites (e.g., Plasmodium) containing this modification on membrane proteins. [10] [11] [12] [13] [14] The natural IgM/IgG antibodies against α-Gal are produced in response to bacteria with this modification in the microbiota. 10 In addition to anti-α-Gal antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms such as B-cell maturation, macrophage response, activation of the complement system, upregulation of pro-inflammatory cytokines through the Toll-like receptor 2 (TLR2)/NF-kB innate immune pathway, and TLRmediated induction of the anti-inflammatory nuclear factor-erythroid 2-related factor 2 signalling pathway. [14] [15] [16] In conjunction, the immune response to α-Gal in animal models has shown protection against infectious diseases without inflammatory responses. 10, [12] [13] [14] 17 Based on these results, we have hypothesized that the immune response to α-Gal may play a role in the person-to-person variability in COVID-19 disease symptoms with a putative protective capacity. 18 First, if the virus contains α-Gal, it would be possible to limit the zoonotic transmission of SARS-CoV-2 by antibody-mediated opsonization. 18 Secondly, boosting α-Gal-mediated protective immune and antiinflammatory responses may contribute to the control of COVID-19 while increasing protection to pathogens with α-Gal on their surface that negatively affect the individual response to SARS-CoV-2. 14, 18 To address this hypothesis, herein 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 antibody titers and alteration of anti-α-Gal antibody isotype composition correlated with COVID-19 severity. These results suggested that the inhibition of the α-Gal-induced immune response translates into more aggressive viremia and severe disease symptoms. Reference values for serum immunoglobulin levels 21 were considered in the analysis of the profile of anti-α-Gal antibody isotypes.

Although this methodology has been previously validated, 22 In the blood cell analysis, the ICU patients showed a higher lymphocytopenia, percentage and neutrophil counts when compared to hospital discharge and hospitalized individuals (p < .001; Figure 1A and Table 1 ). The cellular and biochemical indicators of systemic inflammation, neutrophil-lymphocyte count ratio (NLR), C-reactive protein (CRP), and D-dimer levels were higher in ICU patients when compared to other patients (p < .002; Figure 1A and Table 1 ). 3.2 | Immune response to SARS-CoV-2 increased with severity in COVID-19 patients All COVID-19 symptomatic patients showed both IgA and IgG antibody titers against SARS-CoV-2 ( Figure 1B) . In asymptomatic cases, only IgG antibody titers were determined, and all tested positive ( Figure 1B) . However, only the IgG titers against the SARS-CoV-2

Spike protein significantly increased in accordance with disease symptoms (p = .02; Figure 1B ) with a positive correlation (r s > 0; p = 0; Figure 1B) . These results showed that COVID-19 patients were immunocompetent despite the inflammatory response.

The serum IgA, IgE, IgM and IgG antibody response to α-Gal was characterized in healthy individuals and COVID-19 patients at different disease stages (Figures 2 and 3a) . The calculated anti-α-Gal IgE levels were below (8.3E − 5 to 3.4E − 02 kU/l) the cut-off value of 0.35 kU/l used for the diagnosis of the α-Gal syndrome. A negative correlation was observed for IgE, IgM, and IgG between anti-α-Gal antibody titers and disease severity (r s < 0; p = 0; Figure 3A ). The anti-α-Gal IgA antibody titers did not vary between the different groups (p = .21136; Figure 3A ) nor correlate with disease severity (r s = 0.02; p = .91; Figure 3A ). For anti-α-Gal IgM and IgG antibodies, the titers decreased from healthy to ICU individuals (p < .00001; Figures 2 and 3a) . However, in asymptomatic cases, the anti-α-Gal IgE titers were higher than in healthy individuals and symptomatic COVID-19 patients (p < .000001; Figure 3A ). In COVID-19 patients, the IgE but not IgM and IgG antibody titers were higher in hospitalized patients than in hospital discharge and ICU cases (p < .05; Figure 2 ).

The profile of anti-α-Gal antibody isotypes was qualitatively compared between groups including reference values for serum immunoglobulin levels ( Figure 3B ). The results evidenced that anti-α-Gal Figure 4B ). (Table 1) . B, Serum anti-SARS-CoV-2 IgA, IgG (spike) and IgG (nucleocapsid) antibody levels were determined by ELISA. The patients were grouped as asymptomatic (n = 10), hospital discharge (n = 27), hospitalized (n = 29) and ICU (n = 25). The results were compared between different groups by one-way ANOVA test (p < .05). A Spearman rho (r s ) correlation analysis (p < .05) was conducted between anti-Spike IgG antibody titers and disease severity (2 = asymptomatic, 3 = hospital discharge, 4 = hospitalized, 5 = ICU). ANOVA, analysis of variance; COVID-19, coronavirus disease 2019; CRP, C-reactive protein; ELISA, enzyme-linked immunosorbent assay; ICU, intensive care unit; IgA, immunoglobulin A; NLR, neutrophil-lymphocyte count ratio; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 URRA ET AL. 

Systemic inflammation is associated with changes in the quantity and composition of circulating blood cells and has been identified as the primary basic mechanism resulting in disability and increased mortality in COVID-19. 24 As previously reported, 25, 26 infection.

In addition to the observed negative correlation between anti-α-Gal IgE, IgM, and IgG antibody titers and COVID-19 disease severity, our results showed differences in the profile of anti-α-Gal antibody isotypes in COVID-19 cases that may be associated with different disease stages ( Figure 5 ). These results suggested that higher anti-α-Gal IgE levels in asymptomatic cases may reflect an allergic response mediated by this glycan, which reflects the trade-off associated with the immune response to α-Gal that benefits humans by providing immunity to pathogen infection while increasing the risk of developing allergic reactions to this molecule. 12, 13, 17 In healthy individuals as in hospital discharge cases, the higher representation of anti-α-Gal

IgM and/or IgG antibodies may be associated with a protective response to COVID-19. However, in hospitalized patients, the representation of anti-α-Gal antibody isotypes did not vary, which could reflect the absence of protection. Finally, the higher representation of anti-α-Gal IgA antibodies in ICU patients may be associated with the inflammatory response observed in these cases.

In accordance with these results, it was recently shown in endogenous α-Gal-negative turkeys that treatment with probiotic F I G U R E 3 Serum anti-α-Gal antibody response in COVID-19 asymptomatic and symptomatic cases and healthy controls. A, The IgA, IgE, IgM and IgG anti-α-Gal antibody titers were determined by ELISA. Individuals were grouped as healthy controls (n = 37), asymptomatic (n = 10), hospital discharge (n = 27), hospitalized (n = 29) and ICU (n = 25). The results were compared between different groups by one-way ANOVA test (p < .05). A Spearman rho (r s ) correlation analysis (p < .05) was conducted between anti-α-Gal IgA, IgE, IgM and IgG antibody titers and disease severity (1 = healthy, 2 = asymptomatic, 3 = hospital discharge, 4 = hospitalized, 5 = ICU). B, Profile of anti-α-Gal antibody isotype (shown as percentage of antibody titers) for each group. Reference values for serum immunoglobulin levels were included. Antibody isotypes with highest representation on each group are highlighted in red. ANOVA, analysis of variance; COVID-19, coronavirus disease 2019; ELISA, enzyme-linked immunosorbent assay; ICU, intensive care unit; IgA, immunoglobulin A; α-Gal, Galα1-3Galβ1-(3)4GlcNAc-R bacteria with high α-Gal content results in protection against aspergillosis through reduction by still unknown mechanisms in the pro-inflammatory anti-α-Gal IgA response in the lungs. 29 In Spain, differences have been observed in the number of reported cases per 100 000 people by age and sex, with more females at age 20-59 and males at age 60-89 with a higher mortality in males. 30 In this study, differences in age, but not sex, were observed in symptomatic COVID-19 cases (Table 1) . However, the youngest cases corresponded to ICU patients and healthy control individuals, thus reducing the possible effect of age and sex on the observed F I G U R E 4 Salivary anti-α-Gal antibody response in COVID-19 asymptomatic cases and healthy controls. A, The anti-α-Gal IgA and IgG antibody titers were determined by ELISA and compared in asymptomatic cases between serum and saliva samples by Student′s t-test (p < .05; n = 10). B, The anti-α-Gal IgA antibody titers in saliva were determined by ELISA and compared between asymptomatic COVID-19 cases and healthy individuals by Student′s t-test (p < .05; n = 10). COVID-19, coronavirus disease 2019; ELISA, enzyme-linked immunosorbent assay; ICU, intensive care unit; IgA, immunoglobulin A; α-Gal, Galα1-3Galβ1-(3) 4GlcNAc-R F I G U R E 5 A negative correlation between anti-α-Gal antibody titers and COVID-19 disease severity and differences in the profile of anti-α-Gal antibody isotypes may be associated with different disease stages. Our hypothesis is that the dysbacteriosis observed in COVID-19 patients translates into a reduction in total anti-α-Gal antibody titers and alteration of anti-α-Gal antibody isotype composition due to the reduction in the microbiota of α-Gal-containing commensal bacteria. COVID-19, coronavirus disease 2019; IgA, immunoglobulin A; α-Gal, Galα1-3Galβ1-(3)4GlcNAc-R; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 disease symptoms. Furthermore, currently a clear correlation has not been found between age, sex, and the antibody response to α-Gal. [31] [32] [33] The protective response of anti-α-Gal IgM/IgG antibodies against pathogenic organisms containing this modification on membrane proteins has been well documented. [10] [11] [12] [13] [14] 17, 23 In contrast, IgE antibody response against α-Gal has been associated with the allergy to mammalian meat or α-Gal syndrome and other diseases such as atopy, coronary artery disease and atherosclerosis. 33, [35] [36] [37] In this study and based on anti-α-Gal IgE levels, all individuals were negative for α-Gal syndrome. 38 The anti-α-Gal IgM/IgG antibodies can protect from infection by opsonizing pathogens with α-Gal on their surface. 10 Other immune-mediated mechanisms may be also activated in response to α-Gal, 14-16 which can be activated by SARS-CoV-2 expressing blood type B antigen on their envelope. Although not addressed in this study, these findings prompted us to consider that blood type O individuals could produce antibodies against A and B

antigens that in addition to IgM/IgG antibodies against α-Gal, which cross-react with the structurally similar blood B antigen, 46 could be involved in a polyvalent recognition of the SARS-CoV-2 Spike that may be implicated in the human protection to COVID-19.

In our study, we did not collect information on ABO blood type in COVID-19 patients. However, in a related study with a similar group of patients (n = 73; 9, 40 and 24 hospital discharge, hospitalized and ICU patients, respectively), the results did not show significant differences in ABO blood group distribution. Nevertheless, the results suggest that the ABO blood factor should be considered when evaluating the antibody response to α-Gal. Blood type O individuals, who produce anti-A and anti-B antibodies, can be protected against SARS-CoV-2 particles carrying blood antigens A or B.

However, blood type A and B individuals, who produce either anti-A or anti-B antibodies, would be protected only against SARS-CoV-2 particles carrying antigen A or B, respectively. Therefore, both blood type A and B individuals will be highly susceptible to SARS-CoV-2 particles coming from blood type O individuals because these viral particles do not carry either blood antigen A or B on the envelope. 45 Assuming equal replicative fitness for viruses replicating in cells expressing any of the ABO blood group enzymes, an epidemiological dynamics would be created in which, after a large proportion of the population being exposed and infected by SARS-CoV-2, the frequency of individuals with blood types B and A would be equally represented among COVID-19 patients. In contrast, blood type O individuals would be underrepresented, relative to the frequency of these blood groups in the general population. These predictions were corroborated in a large study (n = 750 000) even after adjusting for age, sex, body mass index, race, ethnicity, and co-morbidities. 47 Therefore, the blood type's effects are not explained by other risk factors including age, sex, race, ethnicity, hypertension, diabetes mellitus, obesity, and cardiovascular and respiratory diseases, 40 which support the immunological considerations discussed above.

Based on the fact that natural antibodies against α-Gal are produced in response to bacteria with this modification in the microbiota, 10 our hypothesis is that the dysbacteriosis observed in COVID-19 patients 34 translates into a reduction in total anti-α-Gal antibody titers and alteration of anti-α-Gal antibody isotype composition due to the reduction in the microbiota of α-Gal-containing commensal bacteria and other still uncharacterized mechanisms ( Figure 5 ). Alternatively, and hypothetically, individuals with higher α-Gal content in the microbiota may be less susceptible to COVID-19. Additionally, the pulmonary microbiota can be affected with the presence of gut bacteria in the lungs. 9 In conclusion, according to these results and previous findings in retrovirus, 48 

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This study was partially supported by the Consejería de Educación, 

The authors declare that there are no conflict of interests. URRA ET AL. | 9

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0001-7383-9649José de la Fuente http://orcid.org/0000-0001-7383-9649