key: cord-0719394-jvc8bhya
authors: Gutiérrez-Bautista, Juan Francisco; Rodriguez-Nicolas, Antonio; Rosales-Castillo, Antonio; López-Ruz, Miguel Ángel; Martín-Casares, Antonia María; Fernández-Rubiales, Alonso; Anderson, Per; Garrido, Federico; Ruiz-Cabello, Francisco; López-Nevot, Miguel Ángel
title: Study of HLA-A, -B, -C, -DRB1 and -DQB1 polymorphisms in COVID-19 patients
date: 2021-08-25
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2021.08.009
sha: 5cf06897a82b95abfa6b66c0e23d92e00680d059
doc_id: 719394
cord_uid: jvc8bhya

Human leukocyte antigen (HLA) plays an important role in immune responses to infections, especially in the development of acquired immunity. Given the high degree of polymorphisms that HLA molecules present, some will be more or less effective in controlling SARS-CoV-2 infection. We wanted to analyze whether certain polymorphisms may be involved in the protection or susceptibility to COVID-19. To do this, we studied the polymorphisms in HLA class I (HLA-A, -B and -C) and II (HLA-DRB1 and HLA-DQB1) molecules in 450 patients who required hospitalization for COVID-19, creating one of the largest HLA-typed patient cohort to date. Our results show that there is no relationship between HLA polymorphisms or haplotypes and susceptibility or protection to COVID-19. Our results may contribute to resolve the contradictory data on the role of HLA polymorphisms in COVID-19 infection.

in the development of acquired immunity. Given the high degree of polymorphisms that HLA 26 molecules present, some will be more or less effective in controlling SARS-CoV-2 infection. We 27 wanted to analyze whether certain polymorphisms may be involved in the protection or susceptibility 28 to COVID-19. To do this, we studied the polymorphisms in HLA class I (HLA-A, -B and -C) and II 29 (HLA-DRB1 and HLA-DQB1) molecules in 450 patients who required hospitalization for COVID-19, 30 creating one of the largest HLA-typed patient cohort to date. Our results show that there is no 31 relationship between HLA polymorphisms or haplotypes and susceptibility or protection to COVID-32 Introduction 36 COVID-19 (Coronavirus Disease 2019) is a respiratory tract infection, caused by the severe acute 37 respiratory syndrome coronavirus 2 (SARS-CoV-2), that can progress to pneumonia, acute respiratory 38 distress syndrome (ARDS), cytokine storm, multiple organ failure, and death. 1 The severity of 39 COVID-19 depends on increased age, and comorbidities such as obesity, arterial hypertension, 40 diabetes, heart disease and respiratory disease. 2,3 There is a great need to study factors that play a 41 relevant role in host defense, with the objective of identifying variables of susceptibility and severity. 42

Of special interest are the human leukocyte antigens (HLA), which are highly polymorphic proteins 43 that play a crucial role in the function of adaptive immunity. HLA presents pathogen-derived peptides 44 on the surface of the infected cell, which are then recognized by specific T lymphocytes, inducing an 45 immune response against the pathogen. 4,5,6,7 The high level of polymorphism found in both HLA class 46 I (HLA-A, -B and -C) and class II (HLA-DP, -DQ, -DR) molecules increases the variety of peptides 47 that can be presented and recognized by the immune system. This is due to variations in their 48 physical/chemical properties that increases or decreases their ability to bind and present certain 49 peptides. Because of this, an allele can be a good or poor presenter of peptides derived from a 50 pathogen. There is evidence of HLA alleles that can play a protective or susceptible role in infections 51 caused by human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and 52 plasmodium. 8,9 53 Each person has a different combination of HLA alleles (haplotypes) that determines their ability to 54 respond to certain pathogens. These haplotypes have been selected throughout evolution due to the 55 selective pressure carried out by pathogens. 10 It has been suggested that the differences observed in 56 the number of cases and severity of COVID-19 between different regions of the world, may in part be 57 due to a skewed distribution of HLA alleles involved in protective immunity against SARS-CoV-2. 58 11,12 while the HLA-B*07:03 and HLA-DRB1*03:01 alleles are related to susceptibility to SARS-CoV-1. 62 13, 14 Therefore, we hypothesize that HLA typing of patients infected by SARS-CoV-2 can help us find 64 alleles that are involved in susceptibility, protection, and poor prognosis to COVID-19. To this end, 65

we have performed a high-resolution HLA typing of 450 confirmed SARS-CoV-2 patients who 66 required hospitalization, comparing their allele and haplotype frequencies with a group of 959 67 representative controls. We believe that such an analysis can generate data that could greatly aid the 68 development of personalized treatments, diagnosis and vaccination. 15 69 J o u r n a l P r e -p r o o f

The current study was performed on 450 hospitalized COVID-19 patients at the Hospital Universitario 72

Virgen de las Nieves, Granada, Spain. The samples were collected from April 2020 to January 2021. 73

The inclusion criteria were the need for hospitalization due to pneumonia or respiratory distress due to 74 COVID-19, diagnosed by a positive SARS-CoV-2 PCR or the presence of SARS-CoV-2-specific IgG 75 antibodies in blood as described below. The characteristics and comorbidities of the patient group can 76 be seen in Table 1 . 77

The patients had pneumonia or respiratory distress and were classified according to severity of the 78 disease in need to enter intensive care unit (ICU) / No ICU; need for Mechanical Ventilation / No 79 Mechanical Ventilation; Deceased / Survivors (Table 1) . 80

The control group (n=959) is made up of healthy blood donors, representative of the Granada area, 81 who were not infected at the time of donation. The average age of the group is 45 ± 5 years and 51% 82 of the members are women. 83

The study was reviewed and approved by the Portal de Ética de la Investigación Biomédica. Junta de 84 Andalucía (Cod. 0766-N-20). The patients/participants provided their written informed consent to 85 participate in this study. 86 

For PCR diagnosis we use the cobas® SARS-CoV-2 assay on a cobas® 6800 system (Roche 100 Molecular Systems, Pleasanton, California, USA). This is a single-well, double-target assay that LABType sequence-specific oligonucleotide typing test (One Lambda, Canoga Park, California, 116 USA). Target DNA was amplified by PCR using sequence-specific primers, followed by hybridization 117 with allele-specific oligodeoxynucleotides coupled with fluorescent phycoerythrin-labelled 118 microspheres. Fluorescence intensity was determined using a LABScan 100 system (Luminex xMAP, 119

Austin, Texas, USA). HLA alleles were assigned using the HLA-Fusion software (One Lambda). 120

Statistical tests for alleles, genotypes and haplotypes were performed using the R (R software; R 123

Foundation for Statistical Computing) package BIGDAWG. 16 Hardy-Weinberg equilibrium (HWE) 124 tests were calculated using PyPop software ver. 0.7.0 (http://www.pypop.org). Frequencies of 125 individual HLA alleles in patients and controls were compared using the χ2-test. Variants with 126 expected counts less than five were combined into a common class (binned) prior to computing the χ2-127

The R package BIGDAWG was also used for amino acid analysis. The software uses the collection of 129 alleles in the input dataset, to retrieve a list of aligned amino acid sequences from the IMGT/HLA 130 Significance levels were corrected by Bonferroni correction for multiplicity of testing by the number 133 of comparisons. A corrected P value of <0.05 was considered statistically significant for all statistical 134 tests. 135

Genotype frequencies of the HLA-A, -B, -C, -DRB1 and -DQB1 loci did not deviate from Hardy-138

Weinberg expectations (Supplementary Table 1) . 139

The comparison of the HLA allele frequencies between the control group and hospitalized COVID-19 140 patients identified several alleles with significantly different frequencies. However, the significances 141 were lost after Bonferroni correction (Supplementary Table 2) . 142

The HLA-A*25:01 and DRB1*11:01 alleles had a significantly higher frequency in the control group 143 without correction (Table 2 ). These alleles could represent possible protective alleles to COVID-19. 144

The alleles HLA-A*66:01, B*40:02, B*55:01, DRB1*14:04 and DQB1*05:03 also presented 145 significant values without correction, being overrepresented in hospitalized COVID-19 patients ( Table  146 2). These alleles could be considered as susceptibility alleles to COVID-19. 147

The haplotype study did not show significant results either, but the extended haplotype HLA-A*29:02, 148 B*44:03, C*16:01, DRB1*07:01 and DQB1*02:02 is of interest, which without correction had a p-149 value of 0.013 (Table 3) . 150 

To address the relationship between HLA and disease severity, we compared the allelic and haplotypic 159 frequencies between the following groups: patients admitted to ICU versus patients that no admitted to 160 ICU; patients that needed mechanical ventilation versus patients that did not require mechanical Bonferroni correction no specific amino acids in the HLA loci A, B, C, DRB1 or DQB1 were found to 177 provide any significant contribution to COVID-19 risk or protection (Supplementary table 3) . 178 

The amino acid analysis was also performed between the different severity groups: : patients admitted 183

to ICU versus patients that no admitted to ICU; patients that needed mechanical ventilation versus 184 patients that did not require mechanical ventilation; deceased patients versus surviving patients; and 185 between the aforementioned groups and controls, not finding significant values after Bonferroni 186 correction (data not shown). 187

Our results suggests that there is no significant correlation between particular HLA alleles/haplotypes 191 and susceptibility or protection against COVID-19, which is in agreement with a previous study. 18 192 However, numerous studies have reported alleles of protection or susceptibility to COVID-19. A study 193 conducted with 82 Chinese patients found that the HLA-C*07:29 and HLA-B*15:27 alleles were more 194 frequently detected in the COVID-19 group than in the control population. 19 can evolve into a cytokine storm. 35 An increase in pro-inflammatory cytokines (IL-1, IL-6, IL-12, 242 TNF- and IFN- 36 , as well as lymphopenia and an increase in the activation of the few remaining 243 lymphocytes have been described in the most severe cases. 37 Likewise, the increased activation of 244 CD8+ T lymphocytes, 38 possibly due to the high amount of cytokines, can cause great damage to the 245 lung epithelium, resulting in to hypoxemia, hypotension and even shock. 35 We hypothesize that an 246 efficient presentation of SARS-CoV-2-derived would increase the cytotoxic CD8+ T cell response, 247 augmenting tissue damage in the lungs and elevating the risk of death in severe COVID-19 patients. In 248 addition, it would produce an increase in the activation of T helper lymphocytes (Th), further 249 promoting the inflammatory state. Patients with more severe disease have been shown to have broader 250 and stronger T cell responses 38 , which may be due to a high viral load due to poor viral control by 251 innate immunity 38 , an insufficient early T cell response or to a better recognition of the virus by 252

However, our results suggest that there is no relationship between COVID-19 and HLA 254 polymorphisms, also supported by supertypes and amino acid analysis, indicating an irrelevant role of 255 HLA in the risk of COVID-19 infection, which is in agreement with recent genome-wide association 256 studies (GWAS). 39 In fact, the GWAS identified a polymorphism in IFNAR2 (Interferon alpha and 257 beta receptor subunit 2) related to Given this, we believe that innate immunity is crucial 258 for viral infection control, with interferon playing an important role. 40 The virus has numerous 259 mechanisms to bypass the innate immune system and block an effective interferon response. 41 (Table 3) . Interestingly, the 277 frequency of this haplotype was significantly (P=0.006 without correction) higher (more that 2-fold) in 278 the ICU patients versus control group. These data may suggest that this haplotype is related to severity 279 and admission to the ICU. The frequency of this haplotype is 6.2% in the Spanish population 45 , and 280 may be one of the explanations to the high incidence of severity and mortality that the country has 281 Acknowledgments: This study is part of the doctoral thesis of Juan Francisco Gutiérrez-Bautista. 305

Programa de doctorado en Biomedicina, University of Granada, Granada, Spain. 306

The authors declare that the research was conducted in the absence of any 307 commercial or financial relationships that could be construed as a potential conflict of interest. 308 J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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