key: cord-0789019-5oi27wnn
authors: Gil-Manso, Sergio; Blanco, Iria Miguens; Motyka, Bruce; Halpin, Anne; Lopez-Esteban, Rocio; Perez-Fernandez, Veronica A.; Carbonell, Diego; López-Fernández, Luis Andrés; West, Lori; Correa-Rocha, Rafael; Pion, Marjorie
title: ABO blood group is involved in the quality of the specific immune response
date: 2021-05-24
journal: bioRxiv
DOI: 10.1101/2021.05.23.445114
sha: 1b83a54b8309a2238d791a7c298ef35fe6ce87ef
doc_id: 789019
cord_uid: 5oi27wnn

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12–305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the Spike (S), Nucleocapsid (N), and Membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that A-group individuals presented significantly higher frequencies of specific CD4+ T-cell responses against Pep-M than O-group individuals. The A-group subjects also needed longer to clear the virus and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors determine the sustainability of the body’s defences, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity. Summary This work describes that cellular responses against SARS-CoV-2 M-protein can be detected after 10 months but were lost against S- and N-proteins. Moreover, the individual factors; ABO-group and age influence the sustainability of the specific humoral and cellular immunity against SARS-CoV-2.

Since December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 52 causing coronavirus disease 2019 (COVID-19), has spread worldwide, triggering various clinical 53 manifestations in infected patients, such as dry cough, fatigue, fever, diarrhoea, and 54 pneumonia . The SARS-CoV-2 pandemic poses a serious health threat to the 55 global population. The most effective way to protect the population would be to achieve 56 widespread anti-SARS-CoV-2 immunity, after either natural infection or vaccination. Strong anti-57 SARS-CoV-2 immunity is crucial for reducing the spread of the virus, and information about the Less is known about long-term cellular protection, which is pivotal for resolving viral infections 71 and developing long-lasting immunity. Positive and promising results have suggested that 72 cellular immunity can be generated during SARS-CoV-2 infection (Bilich et were hospitalized with moderate symptoms (n = 2), none of whom required intensive care unit 96 care (Table 1 ). The subjects' ages ranged from 25 to 62 years (Table 1) . 97

To study the generation of SARS-CoV-2-specific memory T-cells against structural nucleocapside 99 (N), spike (S) and membrane (M) proteins, the intracellular cytokine expression of the donor's 100

PBMCs was analysed after 6 hours of stimulation with peptide pools (Fig. 1A shows the gating 101 strategy for the CD4+ subset). The studied cytokines were intracellular IL-2, IL-4, IL-17A, IFN-γ, 102 and TNF-α, when PBMCs were non-treated (NT) or stimulated with Pep-S, Pep-M and Pep-N, 103 cytomegalovirus (Pep-CMV), or CytoStim as a positive control for cellular activation (Fig. S1 ). The 104 CMV stimulation results represented in all the figures were derived only from CMV-seropositive 105 individuals (n = 21) screened using the IgG anti-CMV ELISA kit. The frequencies of IFN-γ-and 106 TNF-α-producing CD4+ T-cells were significantly higher in stimulated conditions than in NT for 107 all the peptides, demonstrating the presence of SARS-CoV-2-specific cells in almost all the 108 individuals (Figs. 1B and 1C, respectively). However, Pep-N did not induce a significant increase 109 in the frequency of TNF-α-producing CD4+ T-cells (Fig. 1C) . As the frequency of cytokine-110 expressing cells was close to the NT condition in some individuals, we calculated the stimulation 111 index (SI), for each individual, by dividing the frequency of specific T-cell response against 112 peptides pools by the respective response in the NT control. An SI above 2 was considered to 113 indicate a detectable response, while that below 2 corresponded to a lack of response from the 114 individual. We observed that most individuals presented a clear and robust signal after 

In our study, most of the individuals did not have comorbidities, and almost all had mild 164 symptoms (Table 1) . We analysed whether some other factors associated with infection 165 susceptibility, such as age and ABO group, were related to a better response to SARS-CoV-2. A 166 positive correlation was observed between age and the time needed to reach viral clearance 167 was low, we observed that the asymptomatic individuals showed significantly higher levels of 177 IgG anti-A (type III) in the plasma than those with mild symptoms (Fig. 4C) . Additionally, the 178 levels of IgG anti-B (type III and IV) were significantly higher in the O-group individuals with low 179 viral loads, on the day of sample processing (as detected by real-time PCR, high CT), than those 180 with higher viral loads (low CT; Fig. 4D ). 181

It has previously been observed that the severity of symptoms and viral load are associated with 182 immune dysregulation. We observed that O-group individuals showed higher absolute numbers 183 (AbsN) of total lymphocytes (1862 ± 174 cells/µl, mean ± SEM) than those in the A-group (1440 184 ± 93 cells/µl, mean ± SEM; p = 0.0196; Fig. 4E ). This can be explained by the lymphopenia already 185 observed in COVID-19 individuals, even those with mild symptoms. Additionally, the individuals 186 from the O-group seemed to recover the AbsN of total lymphocytes after more than 150 days likely indicating a co-ordinated cellular and humoral immune response in the A group (Fig. 5D) . 216

In summary, the ABO blood group is an essential factor that can influence the time of viral 217 clearance and the intensity of the TNF-α-associated response over time P-PCR+, with the A group 218

showing the highest TNF-α-associated response, as well as a significant decrease in response 219 intensity 10 months post-infection. 220

Discussion 222

In this study, we aimed to evaluate the induction and duration of T-cell specific memory and 223 humoral immunity in individuals who had recovered from SARS-CoV-2 infections with 224 asymptomatic/mild symptoms individuals who represent the great majority of infected subjects. 225

We also studied some susceptibility factors that may be associated with the development of 226 CoV-2 S-protein, we observed that the CD8+ T-cell memory response seemed to be deficient. 233

Whether a robust CD8+ T response might be generated is a worthwhile question, as the CD8+ 234

cytotoxic T-cell response is generally profoundly implicated in viral clearance. It has been shown 235 that COVID-19 subjects present elevated Th2-cytokines (IL-4 or IL-10), which could inhibit the 236 (Table 1) . 304

Whole blood was labelled for surface markers. PBMCs were isolated by density gradient 305 centrifugation. The serum was separated by centrifugation, and the supernatant was stored at 306 Table  310 1. 

Whole blood was labelled for surface markers with the antibodies listed in Supplemental Table  329 1. After surface labelling, red blood cells were lysed using RBC Lysis/Fixation Solution 330 Table 1 ). The cells were then analysed by flow cytometry, 335 using a Gallios cytometer (Beckman Coulter, Nyon, Switzerland). All the cytometry data were 336 analysed using the Kaluza software (Beckman Coulter). The gating strategy applied for the 337 analyses of flow cytometry-acquired data is provided in Fig. 1 and Supplemental Fig. 1 . 338

The 96-well CMV IgG ELISA (Abcam, Cambridge, MA, US) was performed according to the 340 manufacturer's instructions. This ELISA detects human anti-cytomegalovirus IgG. The final 341 interpretation of positivity was determined by a ratio above a threshold value given by the 342 manufacturer: positive (ratio > 11), negative (ratio < 9), or non-defined (ratio 9-11). Quality 343 control was performed, following the manufacturer's instructions, on the day of testing. 344 To detect serum ABO and SARS-CoV-2 antibodies, sera (25-fold dilution) were incubated with 367

Luminex beads for 30 minutes at room temperature, washed, and then incubated with a 50-fold 368 dilution of PE-conjugated goat anti-human IgM or IgG (both from Thermo Fisher, Waltham, MA, 369 US) for 30 minutes at room temperature. Samples were acquired using a FLEXMAP 3D® Luminex 370 system (Toronto, Canada). 371

Data are displayed as means with standard error. The statistical tests used to evaluate the 373 experiments are described within the respective figure legends. Continuous data were tested 374 for normality of distribution, and individual groups were tested by use of the Mann-Whitney U 375 test. Spearman's rho (r) was calculated to assess the correlation between continuous data. 376 the first PCR negative after the infection. Therefore, both dates allow the time for viral 585 clearance to be determined. (B) Correlation between plasma levels of IgG anti-S1 and anti-RBD 586 (anti-SARS-CoV-2 immunoglobulins) and the time between PCR+ and PCRneg. Coloured dotted 587 lines estimated threshold of positivity for anti-SARS-CoV-2 immunoglobulin detection. 588

Correlations were assessed using Spearman's rank correlation; *p < 0.05 was considered 589 significant. Each symbol corresponds to an individual. 590 

Human genetic factors 406 associated with susceptibility to SARS-CoV-2 infection and COVID-19 disease severity

XMAP Cookbook: A collection of 409 methods and protocols for developing multiplex assays with xMAP technology

2020. 414 Decline of Humoral Responses against SARS-CoV-2 Spike in Convalescent Individuals

2021a. Differential kinetics of T cell and antibody responses delineate 419 dominant T cell epitopes in long-term immunity after COVID-19

T cell and antibody kinetics delineate SARS-CoV-2 peptides 423 mediating long-term immune responses in COVID-19 convalescent individuals

Persistent cellular immunity to 427 SARS-CoV-2 infection

2020. Persistent Symptoms in 429 Patients After Acute COVID-19

Immunological 433 memory to SARS-CoV-2 assessed for up to 8 months after infection

LICORNE). 2020. Severe 439 SARS-CoV-2 patients develop a higher specific T-cell response

Host Synthesized Carbohydrate Antigens on Viral Glycoproteins as

Heel" of Viruses Contributing to Anti-Viral Immune Protection

Clinical recurrences of COVID-19 symptoms after recovery: Viral relapse, 447 reinfection or inflammatory rebound?

Management of post-449 acute covid-19 in primary care

2020. 453 Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19

Inhibition of the interaction between the SARS-CoV spike protein and its cellular 457 receptor by anti-histo-blood group antibodies

Negative Clinical Evolution in 460 COVID-19 Patients Is Frequently Accompanied With an Increased Proportion

Undifferentiated Th Cells and a Strong Underrepresentation of the Th1 Subset

Modernizing ABO antibody detection: time for new methodologies to 465 support ABO-incompatible transplantation

2020. A minimal common outcome measure set for COVID-471 19 clinical research

475 ABH-Glycan Microarray Characterizes ABO Subtype Antibodies: Fine Specificity of 476 Immune Tolerance After ABO-Incompatible Transplantation

Chemical Basis for Qualitative and 481 Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for 482 Organ Transplantation

Will SARS-CoV-2 Infection Elicit Long-484

Lasting Protective or Sterilising Immunity? Implications for Vaccine Strategies

SARS-CoV-2-specific T cell immunity in cases 489 of COVID-19 and SARS, and uninfected controls

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Factors related to 499 asymptomatic or severe COVID-19 infection

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Relationship 553 between the ABO Blood Group and the COVID-19 Susceptibility

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A) Correlation between the plasma levels of anti-S1 and anti-RBD (anti-SARS-CoV-2 592 immunoglobulins) and the time between the detection of COVID-19 infection (PCR+) and the 593 time of sample processing

Symbols in the grey zones represent samples with SIs less than 2, 596 indicating non-responding individuals. Correlations were assessed using Spearman's rank 597 correlation; * p < 0.05 was considered significant. Each symbol corresponds to an individual. 598 (C) Stacked bars represent the frequencies of individuals with TNF-α-specific T-cell responses 599 corresponding to SIs ≥ 2 (responding) or < 2 (non-responding) when cells were stimulated by 600 Pep-S (orange

Age and blood groups as factors for viral clearance and absolute numbers of 603 lymphocytes 604 (A) Correlation between the ages of the individuals and numbers of days P-PCR+. Correlations 605 were assessed using Spearman's rank correlation. Each symbol corresponds to an individual. 606 (B) Numbers of days between PCR+ and PCRneg in A-group and O-group individuals

C) Plasma levels of anti-A type III immunoglobulins in mild and asymptomatic 608 COVID-19 individuals. Mann-Whitney U test. (D) Plasma levels of anti-B type III and IV 609 immunoglobulins in individuals presenting high and low CT in the real-time PCR the day of 610 sample processing in mild and asymptomatic COVID-19 individuals

Mann-Whitney U tests. (F) Absolute numbers of lymphocytes in A-and O-group 613 individuals tested 12-150 days P-PCR+ or 150-305 days P-PCR+. Mann-Whitney U tests

Heat map of Spearman correlation coefficients for indicated features in O-group individuals. *p 615 < 0.05 was considered significant

Figure 5: Blood groups as factor for specific CD4+ T-cell response 617 (A) Correlation between SI of TNF-α-producing CD4+ T-cells when stimulated with Pep-S and 618 Pep-M in O-/A-groups and days P-PCR+. Correlations were assessed using Spearman's rank 619 correlation. (B) Frequencies of TNF-α-producing CD4+ T-cells, when stimulated with Pep-M in 620 O-/A-group individuals, tested 12-150 days P-PCR+ or 150-305 days P-PCR+. Mann-Whitney U 621 tests. (C) Correlation between level of anti-RBD immunoglobulins and days P-PCR+

Each symbol corresponds to an 623 individual. (D) Heat map of Spearman correlation coefficients of indicated features in A-group 624 (left panel) and O-group

Table 1: Demographic and clinical characteristics of the COVID-19 convalescent patients 626

* From World Health Organization (WHO) Working Group on the Clinical Characterisation and 627 Management of COVID-19 infection. (infection, 2020) 628 †Date of SARS-CoV-2 negative PCR result was not available for nine patients