key: cord-0993180-qago6lir
authors: Frasca, D.; Reidy, L.; Cray, C.; Diaz, A.; Romero, M.; Kahl, K.; Blomberg, B. B.
title: Effects of obesity on serum levels of SARS-CoV-2-specific antibodies in COVID-19 patients
date: 2020-12-20
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.12.18.20248483
sha: 6dc87a236280f6fce69f962f58d9754b99f3e838
doc_id: 993180
cord_uid: qago6lir

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), cause of COVID-19 (Coronavirus Disease of 2019), represents a significant risk to people living with pre-existing conditions associated with exacerbated inflammatory responses and consequent dysfunctional immunity. In this paper, we have evaluated the effects of obesity, a condition associated with chronic systemic inflammation, on the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. Results have shown that SARS-CoV-2 IgG antibodies are negatively associated with Body Mass Index (BMI) in COVID-19 obese patients, as expected based on the known effects of obesity on humoral immunity. Antibodies in COVID-19 obese patients are also negatively associated with serum levels of pro-inflammatory and metabolic markers of inflammaging and pulmonary inflammation, such as SAA (serum amyloid A protein), CRP (C-reactive protein) and ferritin, but positively associated with NEFA (nonesterified fatty acids). These results altogether could help to identify an inflammatory signature with strong predictive value for immune dysfunction that could be targeted to improve humoral immunity in individuals with obesity as well as with other chronic inflammatory conditions.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), the cause of (Coronavirus Disease of 2019), has been efficiently spreading from human-to-human since the last months of 2019 and has been responsible for mild-to-severe respiratory tract infections. Our knowledge of human immune responses to SARS-CoV-2 infection is limited, and the host factors responsible for disease progression and symptom severity are largely unknown. Recently published data have indicated that chronic low-grade systemic inflammation, inflammaging [1] , is the major cause of the cellular and molecular changes induced by SARS-CoV-2 and is responsible for the highest mortality rates [2] .

Inflammaging has been shown to induce chronic immune activation (IA) associated with impairment of immune cell function, as reviewed in [3] . [4] . Moreover, it has been postulated that during the current pandemic, the production of antibodies to SARS-CoV-2 is critical to limit disease progression and neutralizing antibodies present in plasma from convalescent COVID-19 patients have been shown to induce fast recovery of critically ill patients [5] [6] [7] .

Obesity, like viral infections, induces persistent local and systemic inflammation and chronic IA, contributing to functional impairment of immune cells, and decreased immunity. Obesity and associated inflammation lead to several debilitating chronic diseases such as type-2 diabetes, cancer, atherosclerosis, and inflammatory bowel disease [8] [9] [10] [11] [12] [13] [14] [15] . Therefore, obesity represents an additional risk factor for COVID-19 patients. Also, the prevalence of disease, as well as the occurrence of complications in obese individuals, are increased as compared to lean controls. Indeed, a strong association has been shown between obesity, obesity-associated comorbidities and severe outcomes of COVID-19 [16] .

Body Mass Index (BMI) >30 were more likely to be admitted to acute and critical care compared to individuals with a BMI <30 [17] . Previously, it has been demonstrated that obese patients respond poorly to infections [18] [19] [20] , vaccination [21] [22] [23] , and therapies [24] . The obesity-associated dysregulation of the immune system may also extend the duration and heighten the magnitude of the metabolic stress. It is well known that the obese adipose tissue (AT) is heavily infiltrated with immune cells [25, 26] which fuel local inflammation and exacerbate inflammaging. AT in the thorax and abdominal areas induce secretion of additional pro-inflammatory mediators that can further compromise lung function [27, 28] . The infiltrating immune cells, once activated following SARS-CoV-2 infection, contribute to the release of inflammatory mediators. Another significant health problem is that the AT may be a viral reservoir, playing a crucial role in maintaining local and systemic inflammation, persistent IA, and immune dysfunction [29] .

In this study, we have measured serum levels of SARS-CoV-2 Spike-specific IgG antibodies in lean and obese COVID-19 patients as well as in uninfected controls, using an ELISA test developed and standardized in our laboratory. Results show that higher BMI is associated with a higher infection rate with SARS-CoV-2, measured by serum detection of viral RNA and antibodies. Spike-specific IgG antibodies in obese individuals are negatively associated with BMI and with serum levels of proinflammatory and metabolic markers of inflammaging and pulmonary inflammation. Results obtained could help to design an inflammatory signature with a strong predictive value for immune dysfunction that can be targeted to improve humoral immunity in obese infected individuals.

The test group consisted of 52 individuals who were negative, and 72 individuals tested positive Table 1 . Despite several initial reports indicating that COVID-19 patients were characterized by significant lymphocytopenia [30] , this cohort had normal numbers of total WBC, neutrophils, and lymphocytes, as also recently shown by other groups [31] [32] [33] .

In addition to previously measured RT-PCR and LFD results, we also measured IgG antibodies specific for the SARS-CoV-2 (2019-nCoV) S1+S2 (Spike) recombinant protein, using an ELISA developed and standardized in our laboratory. This ELISA confirmed the results previously obtained with RT-PCR and LFD as all the individuals tested negative or positive by RT-PCR and LFD were also negative or positive in our Spike-specific ELISA, respectively. Results in Table 1 show significantly higher levels of Spike-specific IgG antibodies in positive versus negative individuals in our assay.

Next, we examined the BMI of positive and negative individuals. Results in Fig. 1A show that BMI was higher in positive versus negative individuals (27.7 versus 23.5, respectively), suggesting a higher frequency of infected patients in obese versus lean individuals. In the group of positives, higher BMI was also associated with severe respiratory symptoms (29.1 versus 24.6, respectively), as evaluated at the time of hospital admission (Fig. 1B) . Severe respiratory symptoms included high fever, cough, shortness of breath, hypoxia, as recorded at the time of hospital admission.

We measured pro-inflammatory and metabolic markers associated with inflammation in serum samples from positive and negative individuals. We measured SAA (serum amyloid A protein) [34, 35] , CRP (C-reactive protein) [36] [37] [38] [39] and ferritin [38, 40, 41] . These proteins are markers of pathogen-driven pulmonary inflammation, embolism, and disseminated intravascular coagulation, all characteristics of COVID-19, and therefore predictors of adverse health outcomes. Results in Fig Conversely, and very interestingly, serum levels of NEFA (nonesterified fatty acids) were found lower in positive versus negative individuals and lower in patients with severe respiratory symptoms as compared to those with no symptoms (Fig. 3) . These results could be explained by the recently published findings that the receptor binding domain of the Spike protein is physically and tightly bound to an essential fatty acid, the linoleic acid, leading to a locked Spike conformation and reduced ACE2 interaction, at least in vitro [42] .

When we performed correlation analyses between SAA, CRP, ferritin, and NEFA with BMI in positive individuals, we found, as expected, positive associations with SAA, CRP and ferritin, and negative associations with NEFA (Fig. 4) . Correlations between serum pro-inflammatory and metabolic markers in the positive individuals are shown in Table 2 . Moreover, Spike-specific IgG levels were negatively associated with BMI, SAA, and CRP (Fig. 5, top) , and positively associated with NEFA (Fig. 5,   bottom ).

To our knowledge, this is the first study of immunological and inflammatory profiles of COVID-19 obese patients. Although this is a rapidly evolving research field, and it has already been shown that individuals with a confirmed PCR diagnosis of infection develop antibodies against the Spike protein [43] , how obesity may affect the secretion of SARS-CoV-2-specific IgG is poorly elucidated. Results herein show that serum levels of SARS-CoV-2 IgG antibodies are negatively associated with BMI in COVID-19

patients. This result is consistent with the knowledge that obesity is an inflammatory condition associated with inflammaging [1] and metaflammation [44] both of which are negatively associated with a functional immune system [45] . Another result from the present study is the negative association of SARS-CoV-2

IgG antibodies with markers of pulmonary inflammation (SAA, CRP, ferritin) in our cohort of COVID-19 patients. These are major inflammatory mediators and markers of inflammatory lung injury in patients with catastrophic acute respiratory distress syndrome, which is a primary consequence of COVID-19.

SAA, CRP and ferritin are known to induce a cascade of pro-inflammatory events leading to the secretion of additional markers of inflammation that contribute to the exacerbation of local and systemic inflammation resulting in dysfunctional B cells. In particular, SAA has been shown to induce secretion of several pro-inflammatory mediators by macrophages, such as IL-1β, TNF-α, IL-1RA, IL-8 [46, 47] , and IL-33 [48] , through activation of NF-kB and IRF7. Similar to SAA, CRP also induces secretion of proinflammatory cytokines (IL-6, IL-1β, TNF-α) [49] and chemokines (CCL2, CCL3, CCL4) [50] by monocytes and macrophages, whereas ferritin induces IL-1β and IL-12p70 in macrophages [51] . No direct effects of SAA, CRP and ferritin on B cells have been reported.

Our previously published work has shown that obesity decreases the serum antibody response to the influenza vaccine in young and elderly individuals [21] and increases the secretion of autoimmune antibodies [25, 52, 53] . We have shown that the serum concentration of leptin, the hormone secreted by the AT [54] , correlates with the amount of body fat and BMI [55] . This may be at least one molecular mechanism involved in the reduced B cell function in individuals with obesity, as it induces the secretion of pro-inflammatory cytokines (IL-6/TNF-α) in human peripheral blood B cells through activation of JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathways [56, 57] . Leptin also induces intrinsic B cell inflammation as measured by mRNA expression of several markers associated with immunosenescence [58] . Importantly, the expression of these markers in B cells before in vivo/in vitro stimulation negatively correlates with the same B cells' response after stimulation [59] .

We have previously shown that obesity also increases blood frequencies of the subset of double negative (DN) B cells (CD19+CD27-IgD-) [21, 53] which represents the most inflammatory B cell subset. [63] [64] [65] , Rheumatoid Arthritis [66] , Sjogren's disease [67] , Multiple Sclerosis [68] , Alzheimer's disease [69] , and pemphigus [70] . An increase of DN B cells have also been reported in the blood of COVID-19 patients and associated with anti-viral antibody responses and poor clinical outcomes, as recently shown [71] . DN B cells secrete pro-inflammatory mediators that contribute to dysfunctional humoral responses and pathogenic autoantibodies that, instead of targeting disease-causing viruses, target infected individuals' tissues. Anti-phospholipids, anti-type-I interferons, anti-nuclear antibodies and Rheumatoid Factor have been found in a large percentage of COVID-19 patients and linked to severe disease as All rights reserved. No reuse allowed without permission. preprint (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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.12.18.20248483 doi: medRxiv preprint they may inactivate critical components of the anti-viral response [72] . It is also likely, and important to demonstrate, that the neutralizing antibodies in COVID-19 patients may carry autoimmune specificity, as recently shown for broadly neutralizing antibodies against conserved domains in the influenza hemagglutinin with autoreactivity against tissue antigens previously not identified as autoantigens [73] .

Overall, these findings support that the SARS-CoV-2 infection, similar to influenza, may induce selftolerance breakdown to a variety of autoantigens with self-tolerance breakdown already occurring in obese individuals. It is also likely that tissue failure following dissemination of the virus through the blood can induce cell death and release of intracellular antigens not known as autoantigens, in addition to those already released in the AT for mechanisms like hypoxia and consequent cell death, as we have previously demonstrated [25] . The quality of the antibody response in COVID-19 patients with obesity is extremely relevant for future vaccination campaigns to prevent SARS-CoV-2 infection and COVID-19associated complications in this population that is likely to be among the first to benefit from vaccination.

There are at least two limitations in our study. First, the number of individuals recruited is limited.

We are planning to expand this cohort in the future and with additional markers. Second, this study, although straightforward only shows associations and not mechanisms for the associations. Our future studies will address possibilities after in depth approaches.

Experiments were performed using serum samples isolated from individuals tested negative or positive for SARS-CoV-2 RNA by RT-PCR of nasopharyngeal swab samples and by LFIA using the Healgen LFD with specificity for the SARS-CoV-2 Spike antigen. In total, 52 negative and 72 positive serum samples were collected from both inpatient and outpatient settings and frozen until testing was performed. No additional samples were collected from patients, and samples used in this study were only taken for routine clinical care purposes. Samples were de-identified before use in this study. The demographics, BMI, clinical test results, and clinical characteristics of the participants, as well as their SARS-CoV-2-specific IgG ELISA results, are shown in Table 1 

To examine differences between groups, unpaired Student's t tests (two-tailed) were used. To examine relationships between variables, bivariate Pearson's correlation analyses were performed, using GraphPad Prism version 8 software, which was used to construct all graphs.

All rights reserved. No reuse allowed without permission. preprint (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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.12.18.20248483 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (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 this version posted December 20, 2020. ; preprint (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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.12.18.20248483 doi: medRxiv preprint In bold are indicated the correlations that are significant All rights reserved. No reuse allowed without permission. preprint (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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.12.18.20248483 doi: medRxiv preprint

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