Italian ancestral HLA haplotype predisposing to severe COVID-19 by low spike and high IFNalpha binding affinity

Background/Objectives: Validated association between COVID-19 and the most obvious candidate genes, e.g. HLA, is still missing. A weak association with class I HLA-C*04:01 was found for infection in Sardinians and for severity in another mixed population. Auto-antibodies to interferon type I have been implicated in the severity of COVID-19 in two studies. Method(s): The binding affinity between HLA molecules and SARS-CoV-2 spike protein and IFNalpha subunits was evaluated in silico. The presence of antibodies against one or more of the 12 IFNalpha subunits was evaluated in 160 hospitalized COVID-19 patients. The 10 most frequent haplotypes in the Italian population were tested in 1.997 SARS-CoV-2 infected patients (hospitalized versus not hospitalized). Result(s): The presence of auto-antibodies against at least one IFNalpha subunit was detected in 26% of patients. The haplotype A*24:02-B*35:02-C*04:01-DRB1*11:04-DQB1*03:01 was found to predispose to severity (p = 0.0018;p = 0.07 after Bonferroni correction) in patients <50 years. The haplotype includes alleles able to bind spike with low affinity (i.e. C*04:01 and DRB1*11:04) and IFNalpha with high affinity (i.e. DRB1*11:04). Conclusion(s): One of the 10 most frequent ancestral haplotype of the Italian population predisposes to severity likely reducing both innate immunity through IFNalpha auto-antibodies induction and adaptive immunity through weaker spike protein presentation.

HLA binding-groove motifs are associated with myocarditis induction after Pfizer-BioNTech BNT162b2 vaccination

We found a higher incidence of myocarditis in young males who had received Pfizer-BioNTech BNT162b2 vaccinations as compared with historical controls and unvaccinated individuals. The analyses focused on risk following the first and second vaccine in adults and adolescents, as well as risk in adults following the third (booster) vaccine. Males, mainly aged 12-30 years, were found to be at higher risk. However, the question remains what causes lead one specific young male, but not another, to develop post-vaccination myocarditis. The HLA molecule is known to play an important role in infectious and auto-inflammatory diseases. We hypothesized that differences in HLA alleles could lead to either protection or susceptibility to vaccination-induced myocarditis. On this basis, HLA typing was performed using next-generation sequencing technology for the HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci, in 21 wellcharacterized patients who developed myocarditis after the second Pfizer BNT162b2 vaccination. The HLA genotypes were compared with high-resolution HLA data of 272 healthy controls from the Hadassah Bone Marrow registry samples, who are representative of HLA frequencies in the Israeli population. Our findings demonstrated that in HLA class II, DRB1*14:01 (19.04% vs. 5.3%, Pcorr = 0.028, OR = 4.17), HLA-DQB1*05:03 (19.04% vs. 6.06%, Pcorr = 0.034, OR = 3.64) and DRB1*15:03 (7.14% vs. 0.0%, Pcorr = 0.003, OR = 41.76) were significantly associated with disease susceptibility. We further discovered susceptibility motifs in the HLA-DR peptidebinding grooves: His60 (Pcorr0.01, OR = 3.52) and Arg70 (Pcorr = 0.0047, OR = 3.43). Our findings suggest that immunogenetic fingerprints in HLA peptide-binding grooves may have changed the binding affinity of different peptides derived from the Pfizer-BioNTech BNT162b2 vaccination, and induced myocarditis.

HLA DRB1*15/DRB1*16 might not be protective alleles in COVID-19- induced large vessel giant cell arteritis

Case report Background: Giant cell arteritis (GCA) is an immune-mediated vasculitis affecting large arteries. It has been hypothesized that pathogens including viruses may trigger inflammation within the vessel walls. Human leukocyte antigens' (HLA) genetic studies have previously reported HLA-DR4 (HLA-DRB1* 04 and HLA-DRB1* 01) as susceptibility, and HLA-DR2 (HLA-DRB1* 15 and HLA-DRB1* 16) as protective alleles for GCA. Here we report two cases of large vessel (LV) GCA diagnosed in patients previously suffered from mild coronavirus disese 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2). Case presentation: First case, a 69-year- old male, had a mild COVD-19 three months before the appearance of headache, malaise, and a febrile state associated with extremely increased inflammatory parameters (CRP 2847 mg/dl and IL-6 802.3 pg/ml). Computed tomography examination of the aorta (CTA) and the branches, performed in two occasions six months apart, showed an interesting picture of a migratory arteritis. HLA typing showed: HLA-A* 2,-A* 24;-B* 51,-B* 57;-DRB1* 15,-DRB1* 16;-DQB1* 05,-DQB1* 06;Second case, a 64-year- old female, was evaluated for LV-GCA two months after a mild COVID-19, when she presented with elevated CRP (183mg/dl) and systemic symptoms. Thickening of the ascending aorta and the aortic arch was seen on CTA. Typing of HLA revealed: HLA-A* 2,-A* 11;-B* 27,-B* 35;-DRB1* 14,-DRB1* 15;-DQB1* 05,-DQB1* 06;A whole-body 18F-FDG- PET/ CT performed in both cases revealed inflammation of the ascending, aortic arch, thoracic and abdominal aorta. The first patient had appearance of the inflammatory involvement of the iliac and femoral arteries, while the second patient had an additional pulmonary trunk inflammation. Corticosteroid treatment was introduced in both cases. Due to a progressive inflammatory course of LV-GCA in the first case, the IL-6 inhibitor (tocilizumab) was initiated, leading to a clinical and laboratory improvement. In conclusion, LV-GCA may be considered as an autoimmune disease triggered by SARS-CoV- 2, as one of the broad spectrum of manifestation within the post acute COVID-19. None of the previously known HLA susceptibility alleles for GCA were detected in our patients. In contrast, both patients had DRB1*15 allele, and one of them was DRB1*15/DRB1*16 carrier, suggesting a possibility of losing their protective effect in LV-GCA induced by COVID-19.

Association between HLA and SARSCOV- 2 infection in Mexican Mestizos

The Acute Respiratory Syndrome caused by the new coronavirus described in Wuhan, China in 2019 is a viral, respiratory multifactorial infectious disease, which presents different stages depending on genetic and environmental factors that influence severity. As December 19, 2022, 653,192,573 COVID-19 cases worldwide and over six million deaths;330,795 occurred in Mexico, were reported. Our aim was to analyze the contribution of HLA in Mexican patients infected with COVID-19, categorized in different clinical subgroups. A total of 114 COVID-19 patients and 164 healthy controls, all of them Mexican Mestizos from the highlands, were included in the study;RNA columns were used for extraction, and real-time PCR method was performed for the virus identification. DNA was isolated with the Maxwell16 system and 11 HLA loci were typed using NGS (CareDx, Immucor, and One Lambda). The subjects included: 22 asymptomatic, 86 symptomatic and 109 who were previously vaccinated. We compared controls versus positive patients;versus symptomatic;vaccinated negative versus vaccinated positive;controls versus asymptomatic;asymptomatic versus symptomatic individuals. The significant high risk alleles were A*29:02 (OR = 3.95), B*45:01 (OR = 6.92), C*03:04 (OR = 2.24). DPB1*03:01(OR = 3.17) is a susceptibility marker in vaccinated and unvaccinated patients. The latter is prevalent in Hispanics, Russia, Finland, Spain and the United Kingdom. DQA1*02:01 (p = 0.009, OR = 1.96;DQB1*02:02 (p = 0.009, OR = 2.13) was a susceptibility marker in infected patients who were vaccinated. This is prevalent in Argentina, Brazil, Algeria, Australia, Canada, and China, while high-risk B*45:01 and C*03:04 are prevalent in India, Israel, Eastern Europe, and Mediterranean countries. Protective alleles where DRB1*04:01, A*02:01, DQB1*03:01 and DPB1*02:01. These data are relevant to prioritize vaccination, according to the HLA profile in Mexicans, therefore these data are relevant for the epidemiology of COVID-19.

Role of HLA polymorphism in COVID-19 progression in the Bulgarian population

The SARS-CoV-2 outbreak has impacted different socioeconomic aspects of our societies and represents a global health problem. The different rates of infection are heavily influenced by host genetic factors such as the variability in the HLA region. The aim of our study was to evaluate the role of specific HLA alleles in the Bulgarian population that contribute to COVID-19 progression. 76 Bulgarian patients (median age 59.4;range 25-84) with COVID-19, separated into 3 groups based on the severity of the infection, were included in the study. All patients were typed at allele level for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. Data from a representative Bulgarian population control group of young individuals (n = 540;median age 28,5;range 18-38) with unknown infectious status was used for comparison. Among the patient group, HLA-A*23:01 (OR = 3.16;p = 0.016), and DQB1*05:03 (OR = 3.1;p = 0.0009) showed positive association with moderate course of COVID-19, whereas DRB1*07:01 (OR = 2.54;p = 0.018) was associated with more severe disease. We found that A*01:01 (OR = 1.82;p = 0.018), B*35:03 (OR = 2.22;p = 0.011), B*40:06 (OR = 23.59;p = 0.0001), and DRB1*14:01 (OR = 3.37;p = 0.015) strongly correlate with the disease progression and could be considered as high-risk alleles. Furthermore, A*02:01 (OR = 0.4;p = 0.02), and DQB1*03:01 (OR = 0.47;p = 0.025) were predominantly found in asymptomatic patients and controls and probably exert a protective effect. Population-based and intra-individual variability of the HLA complex could partially explain the different courses of COVID-19. Despite some limitations, our preliminary data shows that some HLA alleles may be associated with a more severe course of the SARS-Cov infection, while others could possibly be considered protective. This work was supported by grant KP06DK1/13, 2021, Ministry of Education and Science, Bulgaria.

Immunogenetics and SARS-CoV-2 infection

Since the beginning of the SARS-Cov-2 pandemic, in 2020, numerous data with respect to the influence of immunogenetics to the predisposition and infection severity have been reported worldwide. It is well accepted that immunogenetics plays a pivotal role in infection and vaccination, as well as vaccination failures and/or breakthrough. Factors of the major histocompatibility complex and the common ABO blood group system have been so far discussed. Here, we describe the association of HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, -DPB1, and HLA-E, -F, -G, -H on the results of molecular detection of COVID-19 or in some cases on antibody detection upon first testing. Furthermore, we defined molecularly 22 blood group systems comprising 26 genes and 5 platelet antigen genes. We observed 37% COVID-19 PCR negative individuals and 63% positive. Within the negative subjects HLA-B*57:01, HLA-B*55:01, DRB1*13:01, DRB1*01:01, were enriched, and in the positive group homozygosity for DQA1/DQB1, DRB1*09:01 and DRB1*15:01. For HLA-DQA1 we observe an enrichment for DQA1*01:01, DQA1*02:01 and DQA1*01:03. For HLADQB1 we found HLA-DQB1*06:02 was enriched in the positive group while HLA-DQB1*05:01 and HLA-DQB1*06:03 in the negative group. We observed a significant enrichment of homozygosity for DQA1/DQB1 in the positive group. The homozygous platelet antigen HPA-1a was significantly enriched in the negative group, contrasting the result of HPA-1ab that was enriched in the COVID-19 infected group. Despite limitations of our study, the data presented here show clearly that COVID-19 infection and all the consequences of that are multifactorial and multigenetic. The virus is in a continuous mutation/selection process leading to escape possibilities. Therefore, associations are a momentum in science.

HLA allele association studies with the kinetics of SARS-CoV-2 spike protein-specific IgG antibody responses to BNT162b2 mRNA vaccine

BNT162b2, an mRNA-based SARS-CoV-2 vaccine (Pfizer- BioNTech), is one of the most effective COVID-19 vaccines and has been approved by more than 130 countries worldwide. However, several studies have reported that the COVID-19 vaccine shows high interpersonal variability in terms of humoral and cellular responses, such as those with respect to SARS-CoV-2 spike protein immunoglobulin (Ig)G, IgA, IgM, neutralizing antibodies, and CD4+ & CD8+ T cells. The objective of this study is to investigate the kinetic changes in anti-SARS-CoV-2 spike IgG (IgG-S) profiles and adverse reactions and their associations with HLA profiles among 100 hospital workers from the Center Hospital of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan. DQA1*03:03:01 (p = 0.017;OR 2.80, 95% CI 1.05-7.25) was significantly associated with higher IgG-S production after two doses of BNT162b2 while DQB1*06:01:01:01 (p = 0.028, OR 0.27, 95% CI 0.05-0.94) was significantly associated with IgG-S declines after two doses of BNT162b2. No HLA alleles were significantly associated with either local symptoms or fever. However, C*12:02:02 (p = 0.058;OR 0.42, 95% CI 0.15-1.16), B*52:01:01 (p = 0.031;OR 0.38, 95% CI 0.14-1.03), DQA1*03:02:01 (p = 0.028;OR 0.39, 95% CI 0.15-1.00) and DPB1*02:01:02 (p = 0.024;OR 0.45, 95% CI 0.21-0.97) appeared significantly associated with protection against systemic symptoms after two doses of BNT162b2 vaccination. Further studies with larger sample sizes are clearly warranted to determine HLA allele associations with the production and long-term sustainability of IgG-S after COVID-19 vaccination.

Impact of HLA diversity on humoral response to SARS-Cov-2 and HBV vaccines in liver transplant recipients

Organ transplant recipients show weaker immune responses to vaccines than immunocompetent individuals, which may be related to the repertoire of HLAbound vaccine antigens presented to T lymphocytes. The HLA evolutionary divergence (HED) metric, which quantifies pairwise allele divergence at each HLA locus, provides a primary measure of the breadth of the immunopeptidome. We recently showed that high class I HED of the donor is a strong and independent driver of allograft rejection in a large cohort of liver transplant recipients. Here, in the same cohort, we explored the relation between HED, the size of the predicted immunopeptidome derived from vaccine antigens, and the quality of vaccine responses. We analyzed humoral response to the SARS-CoV-2 BNT162b2 vaccine (n = 310 patients;undetectable anti-spike IgG titers considered as no response, <=250 BAU/mL as moderate and >250 BAU/mL as strong response) and Hepatitis B virus (HBV) vaccine (n = 424 patients;anti-HBs IgG <10 mIU/mL considered as no response, 10-100 mIU/mL as moderate and >=100 mIU/mL as strong response). HED at HLA-A, -B, -C, -DRB1, -DQA1 and -DQB1 loci were measured using the Grantham distance. NetMHCIIpan-4.0 was used to predict the binding to HLA-DQ molecules of all possible 15mer peptides derived from the Spike and HBS sequences. For each vaccine, HED at the DQB1 locus, but not at the other loci, was significantly higher in responders than in non-responders (p = 0.0003), independent of response-associated covariates (age, time since transplant, immunosuppression). Moreover, for both vaccines, there was a strong relationship between DQB1 HED, the diversity of the immunopeptidome and the quality of the vaccine response. In conclusion, DQB1 HED is a critical determinant of humoral response to vaccines in liver transplant recipients. This metric could guide the design of future vaccines as it predicts the magnitude of the repertoire of vaccine-derived peptides presented to CD4 helper T cells.

An Unusual Case of Autoantibody-Negative KetosisProne Diabetes (KPD) Secondary to Marked Insulin Resistance

KPD is classically regarded as an atypical form of diabetes caused by near-complete beta-cell failure. A 37-year-old Egyptian man (BMI: 27.7 Kg/m2) presented with hyperglycemia (362 mg/dL) and DKA (arterial pH 7.20, ketonemia 5.0 mmol/L, ketonuria 80 mg/dL) . He was afebrile, with recent polyuria, polydipsia and weight loss. HbA1c was 107 mmol/mol (11.9%) and blood tests excluded diabetes secondary to endocrinopathies. SARS-CoV-2 RT-PCR test was negative. IV insulin infusion (0.1 IU/kg/h) and IV fluid therapy were started. He was shortly transitioned to a sc basal-bolus insulin regimen (0.7 IU/kg/day) . Mixed-meal tolerance test (MMTT) revealed a peak 120-min stimulated C-peptide of 12.3 ng/mL, suggesting marked insulin resistance. Islet autoantibodies (ICA, IAA, GADA, IA-2A, ZnT8A) and insulin receptor autoantibodies (IgG/IgM) were negative. HLA genotyping detected the following haplotypes: DRB1∗01, ∗04;DQA1∗01:01P, ∗03:01P;DQB1∗03:02P, ∗05:01P. Insulin dose was gradually reduced and insulin therapy was discontinued after 4 months in favor of metformin (2550 mg/day) plus sc semaglutide (up to 1 mg/week) . After one year, MMTT revealed a peak 60-min stimulated C-peptide of 8.25 ng/mL. During the 18-month follow-up period, fasting capillary beta-hydroxybutyrate values were <0.2 mmol/L and HbA1c remained <48 mmol/mol (<6.5%) , indicating disease remission. This case suggests the existence of an autoantibody-negative KPD subtype driven by marked insulin resistance rather than by insulinopenia.

HLA typing for post-transfused patient: comparison between blood and buccal swab sample

Background: The polymorphic Human Leukocyte Antigens (HLA) play an important role in determining the best matched donor in a haematopoietic stem cell transplant (HSCT). Hence, accurate HLA typing results are crucial to determine the successfulness of a transplant. Most of the patients of hematologic diseases will receive blood transfusion regularly. There is a potential discrepancy result or ambiguous results when a patient already received non-leukoreduced blood component prior to blood sampling for HLA typing. Aims: To determine differences between the HLA typing result from the DNA extracted from blood sample and buccal swab sample. Methods: Blood sample and buccal swab sample were collected from a total of 66 patients with different hematologic diseases and plan to go for haematopoietic stem cell transplant. These patients received at least one pack of red blood cell or platelet between 1 and 14 days prior to blood sample taken. DNA was extracted from all 66 blood samples and 66 buccal swab samples. All samples were typed for six loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 and HLA-DPB1) with Next Generation Sequencing (NGS). NGS was performed in all those samples using the NGSgo-MX6-1 kit. After the library preparation by using NGSgo-LibrX ligation reagents and the NGSgoIndX adapter, it was sequenced in iSeq 100. The results were then analysed with NGSengine software. The results for blood sample and buccal swab sample were then compared. Results: Out of 66 patients, 25 patients received either red blood cell or platelet component prior to blood sample taken for HLA typing. One patient received red blood cell and plasma and the others received red blood cell and platelets before the sample was taken. There are no differences between the HLA typing result from the DNA extracted from blood sample or buccal swab sample for all the 66 patients. The sequencing noise level for both DNA extracted from blood and buccal swab was well separated from the alleles as we can see from the base variation plot. Summary/Conclusions: This preliminary study only focus on the adult patients with hematologic diseases (ALL, AML, CML, MDS, multiple myeloma, DLBCL, hemophagocytic lymphohistiocytosis, NKT Cell lymphoma and severe aplastic anaemia) for the past 1 year. From the findings of this study, blood samples from the patient who underwent blood transfusion can be used for HLA typing instead of using buccal swab samples. This will lower the risk of Covid-19 infection among the healthcare worker who performs the buccal swab sampling from the patient with unknown status of Covid-19 infectivity. However, an extensive study with the appropriate number of samples needed to confirm this finding in the near future.

HLA-association study with more than 16,000 participants shows no heterozygote advantage for COVID-19 and no protective HLAgenotypes

HLA molecules are key restrictive elements to present intracellular antigens for an effective T-cell response against SARS-CoV-2. HLA alleles vary with respect to their potential to present immunogenic viral epitopes and may therefore determine disease severity. Therefore, we set out to investigate the impact of individual HLA genotypes on the severity of SARS-CoV-2 infections. In August 2020 and July 2021, we performed cross-sectional studies among stem cell donors registered with DKMS in Germany. Volunteers registered for stem cell donation represent a comparable healthy subset of the working age population. Available genetic information was linked to self-reported COVID-19 outcome data. Multivariable regression models were fitted to determine the risk of contracting SARS-CoV-2, severe respiratory tract infection and respiratory hospitalization. More than 200,000 registered donors provided informed consent and participated in the study. Their age ranged from 18 to 61 years. Altogether 16,121 participants donors reported a history of COVID-19. Asymptomatic courses were reported by 1428 participants, mild/moderate symptoms by 10,353 participants, severe respiratory infections by 3913 not requiring hospitalization and respiratory hospitalizations by 427 patients. Notably, we did not observe a heterozygote advantage. The risk for severe infections was not statistically different among individuals with or without homozygosity at HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1. Of 84 HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles which were prevalent in more than 400 participants only the presence of HLA-B∗39:01 had significant impact on the risk for respiratory hospitalization (OR 2.23, p = 0.01) at a significance level of 1%. These findings suggest that the HLA genotype is no major factor determining COVID-19 severity. It is therefore possible that the relatively large viral genome of 29.8 kb encodes for abundant epitopes to mount T-cell responses not limited by the HLA genotype.

Whole exome sequencing of MIS-C patients: Analysis of genes involved in inborn errors of type I IFN immunity, hemophagocytic lymphohistiocytosis (HLH), Kawasaki disease (KD) and TLR7 gene

Introduction: After spring 2020, a series of reports from Europe and USA described clusters of children, presenting life-threatening multisystem inflammatory syndrome in children (MIS-C), associated with antecedent exposure to SARS-CoV-2 (1). In patients with life threatening COVID-19 3.5% were found to have inborn errors in type I IFN signalling pathway (2). A case series of 4 young patients with severe COVID-19 reported rare loss-of-function variants in the TLR7 gene associated with impaired type I IFN responses (3). Clinically, MIS-C shares features with secondary hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), which were also associated with possible infectious trigger and might share a common genetic cause (4). Objectives: We analysed whether MIS-C patients have an underlying presence of genetic variants in exomes associated with inborn errors of type I IFN immunity, HLH, KD and presence of variants in TLR7 gene. Methods: Blood was drawn from 17 MIS-C patients upon submission into the hospital, DNA from peripheral blood was isolated and whole exome sequencing was performed. Variants in the following genes were investigated: type I IFN immunity (TLR3, UNC93B1, TRAF3, TBK1, IRF3/9, IRF7, IFNAR1/2, STAT1/2, IKBKG, TRIF), HLH (AP3B1, CD27, FADD, FAS, FASLG, HPLH1, ITK, LYST, MAGT1, MYO5A, NLRC4, PRF1, RAB27A, RECQL4, SH2D1A, STX11, STXBP2, UNC13D, XIAP, TNFRSF9, CDC42), KD (ITPKC, CD40, FCGR2A, BLK, CASP3, TRX-CAT1-7, PGBD1, LTA, TSBP1, HLA-DQB1/2, HLA-DOB, IGHV1-69) and TLR7 genes. Analysis was focused on rare (GnomAD<0.01) exonic or splicing variants. Results: No common genetic denominators were found in analysed genes. Five rare variants were observed in four patients (4/17). According to ACMG classification variants of uncertain significance (VUS) were found in LYST (2), IKBKG (1), IRF3 (1) and NLRC4 (1) in heterozygous genotype. No clinical evidence was found in ClinVar database for any of the variants, except for one variant in LYST (c.3931A>G:p.M1311V) with uncertain significance for Chédiak-Higashi syndrome and medium prediction scores. Variants in LYST (c.5990C>G:p.A1997G), NLRC4 (c.772T>C:p.C258R) and IRF3 (c.325G>C: p.G109R) have high CADD, Mutation Taster, Polyphen and SIFT prediction scores. And IKBKG (c.325C>G:p.L109V) variant had medium prediction scores. Conclusion: Our findings suggest that MIS-C patients do not share a rare loss-of-function variant in type I IFN immunity genes, TLR7 gene or genes associated either with HLH or KD. Despite numerous clinical, immunological and genetic research of the MIS-C patients, the syndromes pathogenesis and etiologic cause remain elusive.